The Impact of Gefitinib on Epidermal Growth Factor Receptor Signaling Pathways in Cancer

The Impact of Gefitinib on Epidermal Growth Factor Receptor Signaling Pathways in Cancer Steven D. Averbuch Abstract The ErbB family of receptor tyrosine kinases, of which the epidermal growth factor receptor (EGFR) is the prototype, is associated with the formation and malignant progression of most of the common solid tumors. These molecules play a key role in a complex network of signal transduction pathways that function in normal development as well as in neoplastic transformation. The EGFR and other family members are therefore promising targets for new anticancer therapies. In normal tissues, EGFR–tyrosine kinase (TK) activity is strictly controlled. However, in tumor cells, there are multiple mechanisms that can lead to increased or inappropriate EGFR-TK activity, including altered expression of EGFR, its ligand, or interacting molecules; decreased deactivation through phosphatases or downregulation; or mutation of the EGFR protein. Novel therapeutic approaches aimed at inhibiting increased EGFR-TK activity include antibodies that block the extracellular ligand-binding site, antibody or ligand fusion proteins that specifically target toxins to the tumor cells, or small-molecule TK inhibitors (TKIs) that act intracellularly to block downstream signal transduction from EGFR. Studies have shown that such blockade can lead to reduced cellular proliferation, inhibition of survival signals, and inhibition of tumor metastasis and angiogenesis. Additionally, some agents, including EGFR antibodies and TKIs such as gefitinib have been demonstrated to be effective against various human solid tumors in preclinical models and have shown activity in advanced non–small-cell lung cancer and other solid tumors. Clinical Lung Cancer, Vol. 5, Suppl. 1, S5-S10, 2003

Key words: Angiogenesis, ErbB receptors, Lung cancer, Phosphatidylinositol-3 kinase, Survival signals, Tumor metastasis, Tyrosine kinase

Introduction

Figure 1

The ErbB receptor tyrosine kinases (TKs) have key roles in fetal development, tissue repair, and disease processes such as neoplastic transformation.1 The 4 members of this family are the epidermal growth factor receptor (EGFR [ie, ErbB1/HER1]), ErbB-2 (ie, HER2/neu), ErbB-3 (ie, HER3), and ErbB-4 (ie, HER4; Figure 1).2 Although all ErbB family members share sequence homology, they differ in important ways. ErbB-2, unlike the other 3 ErbB receptors, has no known ligand and must express its function by pairing with 1 of the ligand-binding family members. ErbB-3 is unique in that it lacks a functional TK domain but also acts by pairing with another family member. The ErbB receptors play a wide variety of roles in diverse cellular processes. The specificity of the response is regulated through the particular ligand binding, the pairing of different Clinical Research, Oncology, AstraZeneca Pharmaceuticals, Wilmington, DE Submitted: May 1, 2003; Revised: Jul 17, 2003; Accepted: Jul 21, 2003 Address for correspondence: Steven D. Averbuch, MD, Clinical Research – Oncology, AstraZeneca Pharmaceuticals LP, 1800 Concord Pike, P.O. Box 15437, Wilmington, DE 19850 Fax: 302-886-4878; e-mail: [email protected]

Structure and Ligand-Binding Properties of Epidermal Growth Factor Receptor Family Receptors EGFR/ ErbB-1/ HER1

Ligands

Ligand Binding

EGF TGF-α HB-EGF Amphiregulin Betacellulin Epiregulin

ErbB-2/ HER2/neu

ErbB-3/ HER3

ErbB-4/ HER4

Unidentified

Heregulin (Neuregulin)

Heregulin HB-EGF Betacellulin

44

36

48

100

Extracellular 82

59

79

100

Intracellular

TK C-terminus

100 P

P

33

P

P

P 24

P

28

P

P

Structure and ligand-binding specificities of the ErbB (ie, HER) family of receptor tyrosine kinases. No ligand has yet been identified for ErbB-2. The numbers indicated denote the degree of homology relative to EGFR. There is 80% homology between the family members within the TK domain, except for ErbB-3, which is kinase inactive. The extracellular ligand-binding domain is less conserved, as is the C-terminus, which interacts with various effector molecules. Abbreviations: EGF = epidermal growth factor; EGFR = epidermal growth factor receptor; HB = heparin-binding; TGF-α = transforming growth factor–α; TK = tyrosine kinase Adapted with permission from Arteaga CL. The epidermal growth factor receptor: from mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol 2001; 19 (suppl 18):32s-40s.

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Impact of Gefitinib on EGFR Pathways of ligand, presence of the receptor at the cell surface, and degradation of the receptor by intracellular endocytosis.8 ExEGF NRG1-β NRG2-α Ligands cessive EGFR-TK activity has been shown to be transforming in vitro, indicating that deregulation of EGFR-TK is a key step in tumor formation.9 There are multiple mecha2 2 2 3 nisms that can lead to in2 4 4 3 1 3 Receptor 1 3 3 4 1 2 4 1 X Dimers creased EGFR-TK activity and 4 X 1 X X X that have been found to occur in different malignancies. Many human tumors have increased or altered expression of EGFR-TK, its ligands, or hetPl3K Shc Cbl Src PLCγ Ras-GAP Effectors erodimer receptor partners.10 Overexpression of EGFR-TK can be caused by gene amplificaMAPK tion, by increased transcription or translation, or by decreased downregulation of mutant reAll 10 of the possible dimeric receptor combinations are shown. Three ligands for this family are shown: EGF, NRG1-β, and NRG2-α. The solid lines indicate high-affinity receptor interactions and the dashed lines indicate low-affinity interactions. Also shown are some ceptors.11 In addition to hetof the downstream effector molecules and pathways that are triggered by the various ligand-receptor dimer combinations.5 erodimerization between ErbB Abbreviations: EGF = epidermal growth factor; MAPK = mitogen-activated protein kinase; NRG1-β = neuregulin 1-β; NRG2-α = neuregulin 2-α; PI3K = phosphatidylinositol-3 kinase; PLCγ = phospholipase Cγ; Ras-GAP = GTPase activating protein family members, the EGFR-TK also interacts with and can be acfamily members in heterodimeric receptors, and ultimately by the tivated by other cell surface molecules, such as G-protein– coupled various downstream effector molecules that are activated.3,4 Of the receptors, cytokine receptors, ion channels, and adhesion mole4 ErbB family members, EGFR has the largest number of known cules.12-14 Decreased phosphatase activity is another mechanism ligands, including epidermal growth factor (EGF), transforming that can increase EGFR-TK signaling.15 Finally, EGFR-TK may growth factor–α (TGF-α), heparin-binding epidermal growth facbe activated in cancer due to mutation of the EGFR gene. The tor (HB-EGF), amphiregulin, betacellulin, and epiregulin. Heregmost common of the EGFR mutations results in EGFRvIII, which ulin and neuregulin are ligands for ErbB-3, whereas heregulin, is a truncated form of EGFR caused by genomic deletion of exons HB-EGF, and betacellulin are ligands for ErbB-4. Although the 2-7 (nucleotides 275-1075, encoding amino acids 6-276).16,17 ErbB receptors generally exist as monomers in the cell membrane, This truncation eliminates most of the extracellular domain of they dimerize with each other upon ligand binding. As all pairwise EGFR. The result is a constitutively active, highly transforming combinations appear to be possible, a total of 10 different hoEGFR-TK enzyme that is not subject to downregulation. The modimers and heterodimers can be generated (Figure 2).5 This EGFRvIII is expressed by > 50% of high-grade gliomas but multiplicity leads to a corresponding diversity of signaling capacihas not yet been found in any normal tissues.17 This mutation 5 ties, further magnified by the particular ligand(s) binding. There is also present in medulloblastomas and in breast cancer, ovarare > 50 intracellular effector molecules that respond to ErbB sigian cancer, and non–small-cell lung cancer (NSCLC) cells.18 naling. Although some (such as Shc) are activated by nearly all Many of the mechanisms that lead to activation of EGFR-TK dimer combinations, others (such as Cbl) respond to a single may occur in the absence of EGFR overexpression alone, sugdimer type.5,6 The different ErbB dimers also have varying capacgesting that therapies targeting the intracellular TK activity ities to stimulate cell proliferation, from zero stimulation with the may hold promise for clinical applications in a broad range of kinase-inactive ErbB-3/ErbB-3 homodimer to > 10-fold stimulatumor types that are driven by increased EGFR-TK–mediated tion with the ErbB-2/ErbB-3 heterodimer.7 In addition to stimusignaling. Expression of EGFR and its ligands is correlated with palating cell proliferation, activation of ErbB signaling can recruit the tient outcomes in a number of human solid cancers. Patients phosphatidylinositol-3 kinase pathway, which interferes with who underwent surgery for head and neck cancer (N = 91) exapoptosis and increases cell survival.7 perienced significantly longer disease-free survival when their Multiple Mechanisms of Epidermal Growth Factor tumors expressed low levels of EGFR and/or TGF-α comReceptor–Tyrosine Kinase Activation in Solid Tumors pared with high levels (P = 0.0001).19 Similarly, 5-year surIn normal cells, the activity of EGFR-TK is tightly regulatvival was 2 to 3 times higher for patients with NSCLC (N = ed at multiple levels, influenced by factors such as availability 131) whose EGFR-positive tumors did not coexpress EGFR Figure 2

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ErbB Signaling Network

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Steven D. Averbuch ligands (EGF or TGF-α; P < 0.05).20 In another study of patients with NSCLC (N = 119), there was no significant difference in 5year survival between patients with EGFRpositive tumors and those with EGFR-negative tumors. However, within the patient group with EGFR-positive tumors, coexpression of HER2 by tumor was associated with a significantly reduced (P < 0.05) 5-year survival rate compared with patients whose tumors were HER2-negative. In contrast, in the EGFR-negative group, there was no difference in the survival of patients with tumors that did or did not express HER2.21 These results suggest that the presence of EGFR ligand or conditions favoring the EGFR/HER2 heterodimer might be particularly potent in driving tumor progression in NSCLC.

Epidermal Growth Factor Receptor–Tyrosine Kinase as a Pivotal Signal in Tumor Cell Growth

Figure 3 Tyrosine Kinase Signaling Pathways

Ras Farnesylation

Anti-EGFR Antibodies

TK Inhibitors

Grb2

PI3K

SH2

P

P

SH2

SH3 SH3

SOS Ras

TK PI3K Akt

γ PLC Akt Inhibitors

Apoptosis

SH2

P

Raf P

SH3 Blockers

SH2 Blockers

Raf1 antisense

MAPKK MAPK Inhibitor

Nucleus

Ras Exchange Inhibitor

MAPK MAPKK Inhibitor

Transcription Factors

Initiate Cell Cycle

Two or more of these agents may eventually be used in combination to inhibit signaling activity in tumors that depend on these pathways for growth.22 Abbreviations: EGFR = epidermal growth factor receptor; MAPK = mitogen-activated protein kinase; MAPKK = MAPK kinase; PI3K = phosphatidylinositol-3 kinase; PLCγ = phospholipase Cγ; SH = Src homology; SOS = son of sevenless; TGF-α = transforming growth factor–α; TK = tyrosine kinase

EGFR-TK has a pivotal role in diverse signal transduction pathways that affect tumor cell proliferation, metastasis, angiogenesis, and apoptosis. Signal transduction pathways are not insulated parallel tracks from the extracellular space to the nucleus. Instead, they branch and interconnect to form a communicating network; EGFR-TK takes part in extensive cross-talk with other cell-surface and intracellular receptors, which mutually influence each other’s activity.5 As more signal transduction inhibitors are developed and tested as single agents, they may eventually be tried in combinations to shut down signal transduction pathways as rapidly and efficiently as possible (Figure 3).22 Some of these combination trials are already being conducted, such as those evaluating gefitinib combined with trastuzumab. Although monoclonal antibodies (MoAbs) against receptors or ligands act extracellularly, most other types of signal transduction inhibitors act intracellularly, including the TKIs, Ras farnesylation inhibitors, Raf1 antisense oligonucleotides, mitogen-activated protein kinase (MAPK) and MAPK kinase inhibitors, Akt inhibitors, and SH2 and SH3 blockers.22

Approaches for Inhibiting Epidermal Growth Factor Receptor–Tyrosine Kinase in Cancer Because of the importance of EGFR-TK activation in transduction of growth signals in tumors, considerable effort has been put into developing cancer treatment approaches that target EGFR-TK. One approach currently being investigated is the use of MoAbs that bind to EGFR outside the cell and block ligand-induced activation of EGFR-TK. Two examples are cetuximab (C225, Erbitux™), a chimeric mouse/human MoAb, and ABX-EGF, a fully humanized MoAb.23 These agents are being investigated in phase II and III trials in advanced solid tumors. In preclinical models, cetuximab has shown antitumor activity in combination with chemotherapy

or radiation therapy, which provided the rationale for clinical trials exploring these treatment combinations. Another approach to targeting EGFR is the design of fusion proteins between a toxin and either an EGFR ligand or an anti-EGFR MoAb. Targeting the toxin to EGFR-expressing cells results in selective cellular uptake of toxin, leading to tumor cell death. Two examples of toxin-containing inhibitors are TGF-α-Pseudomonas exotoxin 40 and EGFR MoAb-Ricin A.24-26 However, this approach has not yet demonstrated activity in clinical studies. A third approach, use of small-molecule EGFR-TK inhibitors (TKIs), is the furthest along in clinical development. The EGFRTKIs compete with adenosine triphosphate (ATP) for binding to the ATP-binding pocket of EGFR-TK, located inside the cell.25 Whereas ligand and antibodies bind to the extracellular portion of EGFR, TKIs bind to the EGFR intracellular domain. The different TKIs that compete for the ATP-binding site of EGFR-TK were derived from a combination of molecular modeling and random screening of libraries of small compounds. Molecular modeling was based on x-ray crystallographic structures of receptor TKs, as well as the structures of natural TKIs such as soybean-derived genistein.25 Although the various synthetic TKIs are surprisingly different from each other (and from ATP), they share a common structure of two 6-member carbon rings (or one 6-member and one 5-member carbon ring) carrying 3 different substituent groups. The high degree of sequence conservation in the TK domains of receptor and nonreceptor TKs has provided the pharmacologic opportunity to synthesize many small-molecule inhibitors that are related but nonetheless show specificity for particular TKs.25 Table 1 lists a number of TKIs of different origins that have varying capacities to inhibit ErbB family receptors.2,25,27 These include the specific, reversible EGFR-TKIs gefitinib and erlotinib (OSI-774, Tarceva™), the irreversible TKIs, and the pan-HER inhibitors, which inhibit EGFR and

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Impact of Gefitinib on EGFR Pathways Table 1

Small-Molecule Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors2,25,27

Small-Molecule Inhibitors

Producer

AG1478 (SU5259)

Sugen

AG1515 (SU5271)

Sugen

CI-1033 (PD183805)*

Pfizer

EKB-569†

Wyeth

GW572016†

Figure 4 Inhibition of Tyrosine Kinase Signaling Pathways

Gefitinib (1 μM) 0

4

6

12

24

Hour EGFR P-Tyr MAPK

GlaxoSmithKline

Gefitinib (ZD1839, Iressa®)‡

Pfizer

PD168393†

Pfizer

Erlotinib (OSI-774, Tarceva™)

P-MAPK

AstraZeneca

PD153035

PKI-166 (CGP75166)

Akt P-Akt

Novartis OSI/Genentech

Cyclin D1

*Irreversible pan-ErbB/HER inhibitor. †Bifunctional (EGFR/HER2) inhibitors. ‡Approved for advanced non–small-cell lung cancer.

HER2 TKs equally well.2,25,27 EGFR-TKIs such as gefitinib can rapidly disable signal transduction pathways downstream of EGFR (Figure 4).2,5 After only 2 hours of incubation of tumor cells derived from various human tumors with gefitinib in vitro, cells contained significantly lower levels of phosphorylated, or activated, EGFR-TK, as well as the activated forms of downstream signaling molecules such as MAPK. Treatment with gefitinib also increased levels of the downstream molecule p27, which correlates with cell cycle growth arrest. In other preclinical studies, gefitinib has been found to block proliferation, migration, and invasiveness of tumor cells, as well as removing the block in the apoptotic pathway that is associated with unregulated EGFR-TK activity.28,29 In a mouse xenograft model, treatment with gefitinib resulted in complete reduction of established human A431 advanced squamous cell tumors (Figure 5).30 Tumor regression was maintained for 90 days, but when gefitinib was discontinued the tumors regrew rapidly. Gefitinib treatment in preclinical tumor models has resulted in additive or synergistic tumor inhibition when this EGFR-TKI was combined with chemotherapy agents or radiation therapy.31-33 For example, in a human colon cancer xenograft model, gefitinib treatment enhanced the antitumor effects of radiation therapy, which was given as either single or fractionated doses (Figure 6).32,33 Blocking EGFR-TK activity in tumors may contribute directly to enhanced radiation sensitivity through cell-cycle alterations and release of the apoptotic block, as well as opposing radiation resistance, which may be directly linked to EGFR-TK activation.34,35 There is also evidence that combined treatment with 2 anti-HER–targeted agents may produce additive antitumor effects. Preclinical studies have shown that gefitinib and the anti-HER2

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2

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p27

Immunoblots from extracts of A431 carcinoma cells during treatment with gefitinib at 1 μM show inhibition of EGFR–tyrosine kinase, as evidenced by lack of autophosphorylation, as well as changes in downstream effectors. These molecular changes are consistent with inhibition of tumor growth by gefitinib. Abbreviations: EGFR = epidermal growth factor receptor; MAPK = mitogen-activated protein kinase; P-MAPK = phosphorylated MAPK; P-Tyr = phosphotyrosine Adapted with permission from C.L. Arteaga, MD.

MoAb trastuzumab each inhibit the growth of BT-474 xenografts, which express EGFR and overexpress HER2. However, the combination of gefitinib and trastuzumab has stronger inhibitory activity than does either agent alone.36

Conclusion Results from clinical trials of EGFR-TKIs are encouraging. In 2 large phase II trials in patients with previously treated advanced NSCLC, gefitinib demonstrated antitumor activity with response rates of approximately 10%-20%.37,38 However, recent results from trials of gefitinib in combination with cytotoxic chemotherapy failed to provide a survival improvement in the first-line treatment of patients with advanced NSCLC.39,40 For the various EGFR-targeted approaches for cancer treatment, a number of questions remain to be answered at both the mechanistic and clinical levels. We need a better understanding of the molecular mechanisms by which these inhibitors act and a better understanding of potential mechanisms of tumor resistance. From these insights, clinical trials will need to address whether response to EGFR-targeted therapies can be predicted based on clinical or tumor characteristics. Surrogate markers of EGFR-TK inactivation in tumors are needed to define the optimal biologic doses of EGFR-TKIs. Although preclinical studies have suggested additive effects when EGFR-TKIs are combined with standard chemotherapy or radiation therapy, data from appropriately designed and well-conducted clinical trials will be re-

Steven D. Averbuch Figure 5 Gefitinib Treatment Resulted in Complete Inhibition of A431 Squamous Cell Tumors

Figure 6 Gefitinib-Enhanced Antitumor Effects of Radiation Therapy 1000

Gefitinib 800 Tumor Volume ( L)

1.2

m

Tumor Volume (cm3)

1.6

0.8 0.4

0

20

40

60

80

100

120

600

400 Single Dose Vehicle Gefitinib Vehicle + 5 Gy Gefitinib + 5 Gy

140

Day

0

10 15 Days After Treatment

20

25

1000 Fractionated Dose Vehicle Gefitinib Vehicle + 3 x 2 Gy Gefitinib + 5 x 2 Gy Gefitinib + 3 x 2 Gy

800

600

400

200

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5

m

quired to establish these effects. Chemoprevention and adjuvant therapy settings are 2 additional applications that should be assessed and compared. Aberrant EGFR-TK signaling is present and may be even more important earlier in the disease process. To explore this potential, an optimal dose for chronic administration must be determined, as well as safety profiles of prolonged inactivation of EGFR-TK in patients with or without cancer. Although many such questions remain to be answered, the new anti–EGFR-TK agents clearly have potential for treatment of common solid cancers.

P = 0.024

Tumor Volume ( L)

Effect of treatment with oral gefitinib at 200 mg/kg per day on human EGFRexpressing A431 squamous tumors grown as mouse xenografts. Tumor-bearing mice were treated on days 29 through 118, at which time gefitinib treatment was withdrawn and vehicle was used on days 119 through 131. Abbreviation: EGFR = epidermal growth factor receptor Adapted with permission from Wakeling AE, et al. ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res 2002; 62:5749-5754.

200

P = 0.024 0

5

10 15 Days After Treatment

20

25

Adapted with permission from Williams KJ, et al. ZD1839 (‘Iressa’), a specific oral epidermal growth factor receptor-tyrosine kinase inhibitor, potentiates radiotherapy in a human colorectal cancer xenograft model. Br J Cancer 2002; 86:1157-1161; and Ranson M. ZD1839 (Iressa™): for more than just nonsmall cell lung cancer. Oncologist 2002; 7(suppl 4):16-24.

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Impact of Gefitinib on EGFR Pathways 21. Tateishi M, Ishida T, Kohdono S, et al. Prognostic influence of the co-expression of epidermal growth factor receptor and c-erbB-2 protein in human lung adenocarcinoma. Surg Oncol 1994; 3:109-113. 22. Levitzki A, Gazit A. Tyrosine kinase inhibition: an approach to drug development. Science 1995; 267:1782-1788. 23. Lynch DH, Yang X-D. Therapeutic potential of ABX-EGF: a fully human anti-epidermal growth factor receptor monoclonal antibody for cancer treatment. Semin Oncol 2002; 29(suppl 4):47-50. 24. Schmidt M, Maurer-Gebhard M, Groner B, et al. Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors. Oncogene 1999; 18:1711-1721. 25. Noonberg SB, Benz CC. Tyrosine kinase inhibitors targeted to the epidermal growth factor receptor subfamily: role as anticancer agents. Drugs 2000; 59:753-767. 26. Raymond E, Faivre S, Armand JP. Epidermal growth factor receptor tyrosine kinase as a target for anticancer therapy. Drugs 2000; 60 (suppl 1):15-23. 27. Ciardiello F. Epidermal growth factor receptor tyrosine kinase inhibitors as anticancer agents. Drugs 2000; 60 (suppl 1):25-32. 28. Chan KC, Knox WF, Gee JM, et al. Effect of epidermal growth factor receptor tyrosine kinase inhibition on epithelial proliferation in normal and premalignant breast. Cancer Res 2002; 62:122-128. 29. Sirotnak FM. Studies with ZD1839 in preclinical models. Semin Oncol 2003; 30(suppl 1):12-20. 30. Wakeling AE, Guy SP, Woodburn JR, et al. ZD1839 (‘Iressa’): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res 2002; 62:5749-5754. 31. Sirotnak FM, Zakowski MF, Miller VA, et al. Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 2000; 6:4885-4892. 32. Williams KJ, Telfer BA, Stratford IJ, et al. ZD1839 (‘Iressa’), a specific oral epider-

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