- Email: [email protected]
Preclinical studies with Erlotinib (Tarceva)
Preclinical Studies With Erlotinib (Tarceva) Robert W. Akita and Mark X. Sliwkowski Erlotinib HCl (Tarceva; Genentech, Inc, South San Francisco, CA) is an orally available, highly selective, reversible inhibitor of epidermal growth factor receptor (HER1/EGFR) tyrosine kinase. Inhibition of tyrosine kinase activity prevents HER1/EGFR phosphorylation, the associated downstream signaling events, and may block tumorigenesis mediated by inappropriate HER1/ EGFR signaling. In vitro and in vivo studies show that erlotinib has activity against human colorectal, head and neck, non–small cell lung, and pancreatic tumor cells. Recent preclinical studies suggest that erlotinib may also have activity against tumors that are dependent on HER2 activation for growth and/or survival. Preclinical studies have addressed the feasibility of using erlotinib in combination with various chemotherapeutic agents, radiotherapy, and targeted agents. Combining agents that have different mechanisms of action has the potential to improve efficacy and inhibit the development of resistance. For example, in preclinical studies, combining erlotinib with cisplatin, doxorubicin, gemcitabine, or low-dose paclitaxel has an additive effect on antitumor activity with no increase in toxicity. Preclinical data provide a strong rationale for investigating erlotinib in the clinical setting. However, additional studies are required to gain further insights into the processes that regulate or influence the antitumor activity of erlotinib. Semin Oncol 30 (suppl 7):15-24. © 2003 Elsevier Inc. All rights reserved.
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HE EPIDERMAL growth factor receptor (HER1/EGFR) is a member of the HER family of receptors. HER1/EGFR is a 170 kd transmembrane glycoprotein that has ligand-dependent intracellular tyrosine kinase (TK) activity. The ligands for HER1/EGFR, which include epidermal growth factor and transforming growth factor-␣ (TGF-␣), bind to HER1/EGFR with similar affinities.1 Ligand binding induces receptor phosphorylation and activates a specific set of cytoplasmic signaling molecules that constitute a complex signaling cascade.2 The mechanism of HER1/EGFR activation and signaling are not fully understood, but data suggest that overexpression and/or dysregulation of HER1/EGFR plays a pivotal role in tumor growth.3,4 Preclinical data show that HER1/ EGFR activation can promote tumorigenesis via a range of processes, including enhanced cellular proliferation, survival, migration, adhesion, and differentiation.2,5 Against this background, HER1/ EGFR was identified as an attractive target for the development of novel anticancer therapies. Several types of agents have been developed that Seminars in Oncology, Vol 30, No 3, Suppl 7 (June), 2003: pp 15-24
target HER1/EGFR. In particular, small-molecule TK inhibitors are recognized as an effective class of receptor antagonists with therapeutic potential. These agents inhibit TK activation and prevent the initiation of downstream signaling events. As a result, they block the cellular effects associated with HER1/EGFR activation.4 ERLOTINIB
Erlotinib HCl (Tarceva; Genentech Inc, South San Francisco, CA) is one of the most promising of a new generation of targeted therapeutics with potential efficacy against a broad range of solid tumors. It is an orally available, quinazoline-based, small-molecule HER1/EGFR-TK inhibitor.6 In vitro studies show that erlotinib inhibits purified HER1/EGFR with an IC50 of 2 nmol/L, while its potency for HER1/EGFR-TK in intact cells is about 10-fold lower.6 Importantly, erlotinib is highly selective for HER1/EGFR and shows minimal activity against other receptor and nonreceptor TKs in vitro. For example, erlotinib has an IC50 of 350 nmol/L for the HER2 TK and greater than 1,000 nmol/L for most other receptor TKs.6 Therefore, erlotinib can block the HER1/EGFR pathway without inhibiting other kinase-dependent pathways that may be essential for normal cellular function. This selectivity has important clinical ramifications for patient tolerability. The affinities of several TK inhibitors for HER1/EGFR and HER2 are shown in Table 1. Erlotinib and gefitinib (Iressa; AstraZeneca, Wilmington, DE), another HER1/EGFR-specific inhibitor, are both in advanced stages of clinical development for the treatment of a variety of tumors. Less specific TK inhibitors, for example, GW-2016 and PKI-166 that inhibit both HER1/EGFR and HER2 (dual inhibitors) and CI-1033 that inhibits all members
From the Department of Molecular Oncology, Genentech, Inc, South San Francisco, CA. Drs Akita and Sliwkowski are employees of Genentech Inc. Address reprint requests to Mark X. Sliwkowski, PhD, Department of Molecular Oncology, Genentech, Inc, South San Francisco, CA, 94080-4990. © 2003 Elsevier Inc. All rights reserved. 0093-7754/03/3003-0703$30.00/0 doi:10.1016/S0093-7754(03)00187-8 15
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Table 1. Affinity of Various Small-Molecule Tyrosine Kinase Inhibitors for HER1/EGFR and HER2
Compound
HER1/EGFR IC50 (mol/L)*
HER2 IC50 (mol/L)
Erlotinib Gefitinib GW-2016 PKI-166 CI-1033 EKB-509
0.02 0.023 0.01 0.025 0.0014 0.039 ⫾ 0.007
0.35 3.7† 0.009 NR 0.009 1.3 ⫾ 0.16
Abbreviations: IC50, 50% inhibitory concentration; NR, not reported. *Concentration required to inhibit 50% of HER1/EGFR in vitro. †Concentration required to inhibit 50% of the HER2 kinase in vitro. Source: Arteaga CL: The epidermal growth factor receptor: From mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol 19:32s-40s, 2001. Reprinted with permission of the American Society of Clinical Oncology.4
of the HER family (pan-HER inhibitor), are in preclinical or early phase clinical development. More data are required to assess the clinical potential of these dual and pan-TK inhibitors. Antitumor Activity Data from in vitro and in vivo preclinical studies show that erlotinib has substantial activity against a variety of tumor types. For example, in vitro studies using DiFi human colon tumor cells show that sub-micromolar concentrations of erlotinib inhibit cell-cycle progression and proliferation and initiate apoptosis.6 In murine xenograft models, orally administered erlotinib blocks ligand-induced HER1/EGFR autophosphorylation and substantially inhibits the growth of human head and neck tumor cells (HN5) with an ED50 of 9.2 mg/kg (Fig 1).7 In mice given a single 92 mg/kg oral dose of erlotinib, maximum HER1/EGFR inhibition was evident after 1 hour, HER1/EGFR autophosphorylation was reduced by 75% to 85% for at least 12 hours, and a significant reduction was observed 24 hours after treatment.7 Erlotinib also caused substantial inhibition of tumor growth in human non–small cell lung cancer (NSCLC) xenograft models developed using H460a or A549 cell lines.8 In the A549 model, tumor growth was
inhibited by 48% and 87% with erlotinib doses of 25 mg/kg and 100 mg/kg, respectively. These in vivo studies clearly show a direct relationship between HER1/EGFR inhibition and cessation of tumor growth. However, further preclinical studies are essential to enable us to fully understand how erlotinib-mediated inhibition of HER1/EGFR TK affects downstream signaling, and how these actions influence antitumor activity. This information should assist in optimizing the clinical application of erlotinib to maximize patient benefit. The effect of erlotinib on human pancreatic carcinoma xenografts implanted in immunodeficient mice has been studied by Ng et al.9 Tumors from two patients with pancreatic cancer were examined; one expressed high HER1/EGFR and moderate HER2 (OCIP#2) and the other expressed moderate HER1/EGFR and HER2 (OCIP#7). Erlotinib significantly inhibited HER1/ EGFR phosphorylation in both xenografts (Fig 2). However, the downstream effects on the phosphatidylinositol 3-kinase, protein kinase B (PI3KPKB/Akt) pathway and the ras-raf-mitogen–activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) pathway were more variable. Erlotinib significantly inhibited the levels of phosphorylated Erk in only one tumor (OCIP#2) and had no significant effect on PKB phosphorylation in either tumor.9 These differential effects may be the result of activation of these downstream effec-
Fig 1. Dose-dependent antitumor response with erlotinib against head and neck tumor xenografts in athymic mice. ED50, effective dose for 50% inhibition of the target receptor. (Modified and reprinted with permission of the American Society for Pharmacology and Experimental Therapies.7)
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Fig 2. Inhibition of HER1/ EGFR phosphorylation (pHER1/ EGFR) in human pancreatic orthotopic xenografts after erlotinib treatment. White bar, without erlotinib (control); striped bar, with erlotinib. (Modified and reprinted with permission of the American Association for Cancer Research.9)
tor molecules through alternative pathways, or possibly the incomplete inhibition of HER1/EGFR signaling by erlotinib. Nevertheless, these findings suggest that erlotinib may benefit patients with pancreatic cancer; a population with few, if any, treatment options beyond surgical intervention. A randomized, double-blind, placebo-controlled phase III clinical trial of erlotinib in combination with gemcitabine in patients with advanced pancreatic cancer (PA.3) is in progress. Erlotinib can also inhibit the growth of cells expressing the HER1/EGFR receptor mutant known as EGFRvIII (also referred to as delta EGFR or de2-7 EGFR). In contrast to wild-type HER1/EGFR, this deletion mutant lacks 267 residues in its extracellular domain, does not bind ligands, and has a constitutively active kinase.10-13 Expression of EGFRvIII has only been reported in tumors and is often associated with a more aggressive phenotype.14,15 In vitro studies show that EGFRvIII-expressing cells are sensitive to erlotinib, although to a lesser extent than cells with wild-type HER1/EGFR.16 These results are consistent with the observation that erlotinib is active against glioblastoma multiforme cells in vitro.17 HER1/EGFR gene amplification occurs in a high proportion of glioblastoma multiforme tumors, and the mutation that encodes EGFRvIII is frequently correlated with this amplification. These prelimi-
nary studies suggest that erlotinib may have utility against gliomas. Additional preclinical and phase I/II studies are in progress to evaluate the therapeutic potential of erlotinib in this indication. The Effect of Other HER Family Members on the Activity of Erlotinib Recent preclinical data show that erlotinib inhibits the growth of both high and modest HER1/ EGFR-expressing tumor cell lines. This observation suggests that HER1/EGFR expression is not the only prerequisite for the antitumor activity of erlotinib.8 Findings from clinical trials are consistent with this observation. They show that response to erlotinib does not appear to correlate with HER1/EGFR expression levels (data on file).18-22 However, unequivocal confirmation of this hypothesis is hindered by the lack of a reliable and reproducible method to quantify HER1/EGFR expression. HER2 is an important member of the HER receptor family. Although it has no known ligand, it potentiates cellular responses to ligand stimulation by forming heterodimers with HER1/EGFR and HER3, and altering their signaling characteristics. This occurs in several ways: association with HER2 increases the affinity of the receptors for their ligands; modulates their turnover rates; and provides a highly active kinase to the receptor
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Fig 3. Relative inhibition of HER1/EGFR and MAPK with erlotinib: MDA-MB-468 cells.24 F, HER1/EGFR; ■, MAPK.
complex.2 The latter effect is particularly significant in the case of HER3 because this receptor lacks intrinsic TK activity and is incapable of signaling in a homodimeric form. Moreover, HER2 is the preferred dimerization partner for HER1/EGFR and HER3.23 In vitro kinase assays show that erlotinib is 20- to 50-fold less effective against HER2 than HER1/EGFR.6 However, because HER2 modulates the signaling characteristics of HER1/EGFR, it is possible that the relatively weak inhibitory activity of erlotinib against the HER2 TK may have a disproportionately large effect on the overall signaling of HER1/HER2 heterodimers. This may, in part, explain the lack of correlation between HER1/EGFR expression and tumor responsiveness to erlotinib in clinical trials (data on file).18-22 In an effort to improve our understanding of the biological activity of erlotinib, dose-response experiments were performed using two HER1/EGFRoverexpressing cell lines (MDA-MB-468 and BT20) stimulated with TGF-␣, and two HER2/ HER3-expressing lines (MCF-7 and MDA-MB453) stimulated with heregulin.24 The results of this study indicate that although increasing doses of erlotinib correlate positively with inhibition of receptor activation and downstream signaling, these effects are not directly proportional.24 For example, the dose–response curves indicate that although 90% of the TGF-␣–stimulated HER1/ EGFR activity in MDA-MB-468 cells is inhibited by 540 nmol/L erlotinib, only 33% of the MAPK activity is blocked at that concentration (Fig 3).
MDA-MB-468 cells express approximately 2 ⫻ 106 HER1/EGFRs per cell.25 Therefore, the 200,000 HER1/EGFRs still capable of signaling after treatment with 540 nmol/L erlotinib can stimulate 67% of the maximum MAPK activity. These data imply that activation of only a small fraction of the HER1/EGFRs is sufficient to generate a substantial mitogenic signal in cells that highly overexpress the receptor. Effective inhibition of signaling by HER1/EGFR-targeted agents may only occur at very high levels of receptor inhibition. Therefore, it is reasonable to propose that HER1/EGFR-targeted agents should be used at doses that are as high as possible (ie, the maximum tolerated dose) to give the best chance of effectively inhibiting not only receptor activation, but also downstream signaling. Erlotinib-mediated inhibition of downstream signaling and receptor phosphorylation are also disproportionate in HER2-expressing cells; however, the relationship is quite different. The same 540 nmol/L concentration of erlotinib inhibits only 28% of the heregulin-stimulated HER2 activity in MCF-7 cells, but blocks almost 90% of the MAPK activity (Fig 4). The extent to which erlotinib inhibits receptor phosphorylation in heregulin-stimulated HER2/HER3-expressing cells was approximately 50-fold lower than for TGF-␣– stimulated receptor phosphorylation in HER1/ EGFR-expressing cells.24 However, the IC50 for erlotinib-mediated inhibition of p42 and p44 MAPK phosphorylation was within a similar range in both cases: approximately 200 nmol/L for
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Fig 4. Relative inhibition of HER2 and MAPK with erlotinib: MCF-7 cells.24 F, HER2/3; ■, MAPK.
heregulin-stimulated HER2/HER3-expressing cells and 500 to 700 nmol/L for TGF-␣–stimulated HER1/EGFR expressing cells. In summary, these findings show that, despite the much lower potency of erlotinib against the HER2 kinase in vitro, it blocks ligand-mediated signaling of HER1/ EGFR and HER2/HER3 with similar potencies. These in vitro results suggest that erlotinib may also be effective against tumors that are dependent on HER2 activation for growth and/or survival. This may increase the range of potential patients that could benefit from treatment with this inhibitor. Studies examining the effects of other HER1/ EGFR inhibitors on tumors that coexpress HER1/ EGFR and HER2 show findings similar to those with erlotinib. In a recent report, treatment with gefitinib resulted in a significant dose-dependent reduction in the level of HER1/EGFR and HER2 tyrosine phosphorylation. Phosphorylation of MAPK and Akt were also reduced.26 In another study, Moulder et al27 found that treatment with 1 mol/L gefitinib substantially inhibited HER2-TK phosphorylation. Finally, AG1478, another quinazoline derivative that inhibits HER1/ EGFR, suppressed the growth of mammary tumors and blocked HER2 phosphorylation in MMTV/ TGF ⫻ MMTV/neu (the mouse homologue of human HER2) bigenic mice.28 Taken together, these data suggest that HER1/EGFR inhibitors could be used to treat patients with solid tumors that express HER1/EGFR and HER2. HER3 and HER4 are also involved in ligand-induced het-
erodimerizations with HER1/EGFR, but the effects of erlotinib on the activation and signaling of these receptor combinations has not been examined in detail. ERLOTINIB IN COMBINATION WITH CONVENTIONAL ANTICANCER THERAPIES
There is evidence to suggest that HER1/EGFRdriven tumor cells, particularly those in which autocrine activation may play a role, can become resistant to chemotherapeutic agents or antihormonal therapy.29 In breast and prostate tumors, the development of an autocrine loop is thought to be a pivotal step in the progression to a hormone-independent state.30,31 Also, a substantial amount of preclinical data from studies with various agents shows that blocking HER1/EGFR activity inhibits the ability of tumor cells to repair the damage caused by chemotherapy or radiation.4,29,32 Therefore, HER1/EGFR overexpression and/or dysregulation may affect the success of many conventional cancer therapies. It is common clinical practice to combine various chemotherapeutic agents and radiotherapy because this approach often improves efficacy and decreases the chance of resistance.33 Against this background, there is a strong rationale to examine the effectiveness of combining erlotinib with chemotherapeutic agents, radiotherapy, and/or antihormonal therapy.
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Table 2. Erlotinib in Combination With Various Chemotherapeutic Agents Combinations With Erlotinib Agent Cisplatin Doxorubicin HCl 5-fluorouracil Paclitaxel Vinorelbine tartrate Gemcitabine HCl
Dose/Route/Regimen 10 mg/kg IV daily ⫻ 1 15 mg/kg IV daily ⫻ 1 200 mg/kg IP daily ⫻ 1 10 mg/kg IP daily ⫻ 5 20 mg/kg IP daily ⫻ 5 25 mg/kg IV daily ⫻ 3 100 mg/kg IP three times a day ⫻ 4
Lethality No No No No No No No
increase increase increase increase increase increase increase
Efficacy Additive effects, no antagonism Additive effects, no antagonism No interaction, no anatagonism Additive effects, no anatagonism No interaction, no antagonism No interaction, no antagonism Additive effects, no antagonism
Abbreviations: IV, intravenous; IP, intraperitoneal. Data on file, Genentech, Inc and OSI Pharmaceuticals, Inc, 2002.
Combination With Chemotherapy The effect of combining erlotinib with several commonly used chemotherapeutic agents was examined in human tumor xenograft models. The results are summarized in Table 2. In all studies, erlotinib was administered at the ED50 (9.2 mg/kg/ day).7 No antagonism of the therapeutic efficacy of any chemotherapeutic agent was observed. Erlotinib in combination with cisplatin, doxorubicin, or gemcitabine had an additive effect on antitumor activity. Additive effects were also observed with paclitaxel at 10 mg/kg, but not 20 mg/kg (data on file).7 Importantly, the toxicities of these chemotherapeutic agents, and the toxicity of erlotinib itself, were not exacerbated in any combination. These data strongly suggest that using erlotinib in combination with certain chemotherapeutic agents may substantially enhance antitumor activity without adversely affecting toxicity. Consequently, clinical trials have been initiated to examine the feasibility and efficacy of several of these combinations. The findings from these preclinical studies also show that the overall antitumor activity achieved when agents are combined is not dependent on the dosing sequence (Fig 5).7 There is no clear evidence to suggest that the sequence of administration is important when erlotinib is combined with these chemotherapeutic agents. However, this may not be the case for all combinations and indications. Additional preclinical studies are in progress to elucidate the mechanism of these additive effects. Phase Ib trials examining erlotinib in combina-
tion with temozolomide; docetaxel; capecitabine; gemcitabine; gemcitabine and cisplatin; carboplatin and paclitaxel; capecitabine and docetaxel; 5-fluorouracil, leucovorin, and oxaliplatin; and docetaxel and carboplatin are currently underway in patients with various advanced solid tumors. Preliminary data from studies of erlotinib in combination with docetaxel,34 gemcitabine and cisplatin,35 and carboplatin and paclitaxel36 are encouraging, showing evidence of antitumor activity and no significant adverse pharmacokinetic interactions between the agents. Importantly, two randomized, double-blind, placebo-controlled phase III clinical trials, TRIBUTE and TALENT, are in progress examining erlotinib in combination with standard chemotherapy as first-line therapy for patients with advanced NSCLC. Combination With Radiotherapy Preclinical data have shown that erlotinib can enhance the effects of radiation in H460 NSCLC cells and H460 NSCLC xenografts implanted in mice.37 The precise mechanism that causes this sensitization to radiation is unknown, but it is proposed that a combination of HER1/EGFR inhibition and downregulation of COX-2 expression are involved. Based on these data, trials of erlotinib in combination with radiotherapy are planned. Several other studies show that other HER1/EGFR inhibitors can also enhance the antiproliferative effects of radiation in vitro and in vivo.38
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Fig 5. Erlotinib in combination with cisplatin: The effect of scheduling. i.v., intravenous; i.p., intraperitoneal; q.d./q.i.d., once daily; t.i.d., three times daily. (Modified and reprinted with permission of the American Society for Pharmacology and Experimental Therapies.7)
Combination With Antiestrogen Therapy There is increasing evidence to suggest that HER1/EGFR signaling pathways are critical for the hormone independence of estrogen receptorpositive breast cancers.39 For example, in vitro studies show that resistance to tamoxifen is associated with upregulation of HER1/EGFR and its signaling pathway.40 Consequently, HER1/EGFR inhibitors could potentially be used to treat antihormone-resistant breast tumors. Preclinical studies show that erlotinib combined with tamoxifen has an additive effect against estrogen receptorpositive HER2-overexpressing human breast cancer cell lines (MDA-361 and MCF-7/HER2; C. Arteaga, personal communication, September 2002). Also, Gee et al41 showed that gefitinib effectively inhibits tamoxifen-resistant breast cancer cell growth in vitro. In view of these data, phase II trials examining erlotinib in combination with letrozole (Femara; Novartis, Basel, Switzerland), a reversible, nonsteroidal aromatase inhibitor, are planned. ERLOTINIB IN COMBINATION WITH TARGETED AGENTS
The regulation of tumor growth and progression is complex, involving many receptors, intracellular
pathways, and genes. Inhibiting more than one crucial pathway or process could be an effective clinical strategy. The advent of novel targeted agents that inhibit receptors and pathways involved in tumorigenesis offer many possibilities for combining various agents with the aim of improving overall therapeutic efficacy. Preclinical studies have a vital role in identifying the most effective combinations before designing clinical trials. These studies are still at an early stage. HER2 Inhibitors Because HER2 is a critical element in the HER signaling network, inhibiting HER2 as well as HER1/EGFR is an attractive proposition. Studies on transfected fibroblasts and transgenic mice show that the growth of tumors induced by excessive HER1/EGFR signaling can be accelerated by the concomitant overexpression of neu (the mouse homologue of HER2).42,43 In vivo studies show that a higher concentration of TK inhibitor is required to inhibit HER1/EGFR phosphorylation in tumors that express both HER1/EGFR and HER2, as opposed to HER1/EGFR alone.44 Moreover, clinical studies in breast and NSCLC show that overexpression of both receptors correlates
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with poorer overall survival compared with overexpression of HER1/EGFR alone.45,46 Preclinical studies of various HER1/EGFR inhibitors and trastuzumab (Herceptin; Genentech, Inc), an anti-HER2 humanized monoclonal antibody, show that HER1/EGFR inhibitors can interfere with HER1/HER2 crosstalk and downregulate HER2 activity, although the exact mechanisms are not fully understood. For example, the addition of gefitinib and trastuzumab to the breast cancer cell line SK-BR-3 resulted in a greater inhibition of cell growth than with either agent alone.26 Preliminary evidence suggests a similar outcome when erlotinib is combined with trastuzumab (C. Arteaga, personal communication, September 2002). Therefore, combining a HER1/EGFR inhibitor, such as erlotinib, with trastuzumab in the clinical setting could have a synergistic inhibitory effect on tumorigenesis, resulting in improved antitumor effects. 2C4 (pertuzumab) is a humanized monoclonal antibody that blocks the association of HER2 with other HER receptors and prevents ligand-dependent and ligand-independent HER2 signaling.47 This is in contrast to trastuzumab, which acts only against the ligand-independent receptor activation associated with HER2 overexpressing tumors. Thus, 2C4 may have activity in patients that would not be candidates for trastuzumab treatment. Its unique mechanism of action suggests that 2C4 has the potential to be highly effective alone and in combination with HER1/EGFR-targeted agents. The effects of combinations of 2C4 and erlotinib on the growth of MDA-MB-175 breast tumor cell lines were examined in a recent study.48 Suboptimal concentrations of both drugs used together resulted in greater caspase 3/7 activation than the same concentration of either agent alone. These data suggest that combined treatment with erlotinib and 2C4 may be efficacious against tumors that do not respond well to single-agent erlotinib. Phase I and II clinical trials of 2C4 monotherapy in various tumors are in progress. Vascular Endothelial Growth Factor-Targeted Agents The HER1/EGFR pathway is involved, to a certain extent, in the production of angiogenic factors such as vascular endothelial growth factor (VEGF).49 Several studies show that HER1/ EGFR-targeted agents can downregulate VEGF
AKITA AND SLIWKOWSKI
production and angiogenesis in human cancer cells.50,51 Combining HER1/EGFR inhibitors with VEGF/angiogenesis inhibitors is an attractive strategy. The potentially complimentary mechanisms of action of these inhibitors may have additive effects on angiogenesis and other VEGF-related actions. These effects could result in a substantial inhibition of tumor growth. To this end, a phase I clinical trial of erlotinib in combination with bevacizumab (Avastin; Genentech Inc), an anti-VEGF monoclonal antibody, for advanced, refractory NSCLC is in progress. CONCLUSION
The biology and genetics underlying the development and progression of solid tumors are complex and involve many different signal transduction pathways. Over the past two decades, however, it has become clear that members of the HER family of growth factor receptors play central roles in the initiation or maintenance of a number of different cancers. Consequently, these receptors have become attractive therapeutic targets. Several agents that specifically target either the HER1/EGFR or HER2 receptors have recently been developed. Findings thus far support the use of these agents in combination with traditional chemotherapy, radiotherapy, and targeted drugs. These new agents are currently being evaluated in combination with existing therapeutic regimens in the treatment of patients with advanced refractory disease. Interpretation of the results from these clinical trials is complicated by the fact that the efficacy of these drugs often does not correlate with the level of expression of their target receptors. This is likely a reflection of the complexity of the signaling network and the fact that the signaling of one HER family member can be modified by heterodimerization with a different member. Preclinical studies are vital for identifying the most important signaling pathways and for elucidating how the interactions of the various HER receptors affect these pathways. These studies promise to improve the probability of positive clinical trial outcomes by allowing for more rational, signaling pathway-based trial designs. This review highlights findings from recent preclinical studies that provide a new perspective on the relationship between erlotinib and the HER signaling network. More studies to examine the actions and interactions of HER1/EGFR and other
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members of the HER family are essential to enable us to fully understand the mechanism of erlotinib action. Results from these studies will hopefully lead to more effective clinical applications of this promising new therapeutic agent. REFERENCES 1. Jones JT, Akita RW, Sliwkowski MX: Binding specificities and affinities of EGF domains for ErbB receptors. FEBS Lett 447:227-231, 1999 2. Yarden Y, Sliwkowski MX: Untangling the ErbB signaling network. Nat Rev Mol Cell Biol 2:127-137, 2001 3. Salomon DS, Brandt R, Ciardiello F, et al: Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 19:183-232, 1995 4. Arteaga CL: The epidermal growth factor receptor: From mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol 19:32S-40S, 2001 5. Wells A: EGF receptor. Int J Biochem Cell Biol 31:637643, 1999 6. Moyer JD, Barbacci EG, Iwata KK, et al: Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res 57:4838-4848, 1997 7. Pollack VA, Savage DM, Baker DA, et al: Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: Dynamics of receptor inhibition in situ and antitumor effects in athymic mice. J Pharmacol Exp Ther 291:739-748, 1999 8. Desai B, Higgins B, Smith M: Antitumor activity of the EGFR/TK inhibitor Tarceva (erlotinib, OSI-774) tumor models. Eur J Cancer 38:63, 2002 (suppl 7) (abstr 203) 9. Ng SS, Tsao MS, Nicklee T, et al: Effects of the epidermal growth factor receptor inhibitor OSI-774, Tarceva, on downstream signaling pathways and apoptosis in human pancreatic adenocarcinoma. Mol Cancer Ther 1:777-783, 2002 10. Ekstrand AJ, Longo N, Hamid ML, et al: Functional characterization of an EGF receptor with a truncated extracellular domain expressed in glioblastomas with EGFR gene amplification. Oncogene 9:2313-2320, 1994 11. Nishikawa R, Ji XD, Harmon RC, et al: A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci U S A 91:7727-7731, 1994 12. Moscatello DK, Montgomery RB, Sundareshan P, et al: Transformational and altered signal transduction by a naturally occurring mutant EGF receptor. Oncogene 13:85-96, 1996 13. Prigent SA, Nagane M, Lin H, et al: Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the RasShc-Grb2 pathway. J Biol Chem 271:25639-25645, 1996 14. Lal A, Glazer CA, Martinson HM, et al: Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion. Cancer Res 62:3335-3339, 2002 15. Damstrup L, Wandahl Pedersen M, Bastholm L, et al: Epidermal growth factor receptor mutation type III transfected into a small cell lung cancer cell line is predominantly localized at the cell surface and enhances the malignant phenotype. Int J Cancer 97:7-14, 2002
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16. Iwata KK, Provoncha K, Gibson N: Inhibition of mutant EGFRvIII transformed cells by tyrosine kinase inhibitor OSI774 (Tarceva). Proc Am Soc Clin Oncol 21:21a, 2002 (abstr 79) 17. Wong AJ, Ruppert JM, Bigner SH, et al: Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc Natl Acad Sci U S A 89:2965-2969, 1992 18. Pe´ rez-Soler R, Chachoua A, Huberman M, et al: A phase II trial of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor OSI-774, following platinum-based chemotherapy, in patients (pts) with advanced-EGFR-expressing, non–small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 20:310a, 2001 (abstr 1235) 19. Pe´ rez-Soler R, Chachoua A, Huberman M. , et al: Determinants of tumor response and survival in patients with relapsing NSCLC treated with Tarceva (erlotinib HCl; OSI774). Final report of a phase II study. Presented at the American Society of Clinical Oncology and Molecular Therapy Symposium, San Diego, CA, November 8-10, 2002 20. Finkler N, Gordon A, Crozier M, et al: Phase 2 evaluation of OSI-774, a potent oral antagonist of the EGFR-TK in patients with advanced ovarian carcinoma. Proc Am Soc Clin Oncol 20:208a, 2001 (abstr 831) 21. Senzer NN, Soulieres D, Siu L, et al: Phase 2 evaluation of OSI-774, a potent oral antagonist of the EGFR-TK in patients with advanced squamous cell carcinoma of the head and neck. Proc Am Soc Clin Oncol 20:2a, 2001 (abstr 6) 22. Hidalgo M: Phase I studies/combination therapy with Tarceva (erlotinib HCl;OSI-774) in head and neck cancer. Presented at the First Annual Opinion Leader Consortium on Novel and Targeted Therapies for Head and Neck Cancer, San Juan, Puerto Rico, February 5-9, 2003 23. Graus-Porta D, Beerli RR, Daly JM, et al: ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 16:1647-1655, 1997 24. Akita RW, Dugger D, Lewis Phillips G, et al: Effects of the EGFR inhibitor Tarceva on activated ErbB2. Proc Am Assoc Cancer Res 43:1003, 2002 (abstr 4973) 25. Kraus MH, Popescu NC, Amsbaugh SC, et al: Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. EMBO J 6:605-610, 1987 26. Normanno N, Campiglio M, De Luca A, et al: Cooperative inhibitory effect of ZD1839 (Iressa) in combination with trastuzumab (Herceptin) on human breast cancer cell growth. Ann Oncol 13:65-72, 2002 27. Moulder SL, Yakes FM, Muthuswamy SK, et al: Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res 61:88878895, 2001 28. Lenferink AE, Simpson JF, Shawver LK, et al: Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu ⫹ MMTV/TGF-alpha bigenic mice. Proc Natl Acad Sci U S A 97:9609-9614, 2000 29. Woodburn JR: The epidermal growth factor receptor and its inhibition in cancer therapy. Pharmacol Ther 82:241250, 1999 30. De Miguel P, Royuela M, Bethencourt R, et al: Immunohistochemical comparative analysis of transforming growth factor alpha, epidermal growth factor, and epidermal growth
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factor receptor in normal, hyperplastic and neoplastic human prostates. Cytokine 11:722-727, 1999 31. Normanno N, Kim N, Wen D, et al: Expression of messenger RNA for amphiregulin, heregulin, and cripto-1, three new members of the epidermal growth factor family, in human breast carcinomas. Breast Cancer Res Treat 35:293-297, 1995 32. Etessami A, Bourhis J: Cetuximab. Anti-EGFR monoclonal antibody. Drugs Fut 25:895-899, 2000 33. Chen X, Yeung TK, Wang Z: Enhanced drug resistance in cells coexpressing ErbB2 with EGF receptor or ErbB3. Biochem Biophys Res Commun 277:757-763, 2000 34. Mita A, Farouzesh B, Hidalgo M: Phase I, pharmacokinetic (PK), and biological studies of the epidermal growth factor receptor-tyrosine kinase (HER1/EGFR-TK) inhibitor erlotinib (OSI-774; Tarceva) in combination with docetaxel. Eur J Cancer 38:53, 2002 (suppl 7) (abstr 168) 35. Ratain MJ, George CM, Janisch L, et al: Phase I trial of erlotinib (OSI-774) in combination with gemcitabine (G) and cisplatin (P) in patients with advanced solid tumors. Proc Am Soc Clin Oncol 21:76b, 2002 (abstr 2115) 36. Patnaik A, Goetz A, Hammond L, et al: Phase I, pharmacokinetic (PK) and biologic study of OSI-774 (Tarceva), a selective epidermal growth factor receptor (EGFR) tyrsoine kinase (TK) inhibitor in combination with paclitaxel and carboplatin in patients with advanced solid malignancies. Eur J Cancer 38:54, 2002 (suppl 7) (abstr 169) 37. Wu H, Kim J, Cao Q, et al: Combined modality therapy of NCI-H460 human lung cancer using epidermal growth factor receptor inhibition, OSI-774 and ionizing radiation in vitro and in vivo. Int J Radiat Oncol 54:176, 2002 (suppl) (abstr 1031) 38. Harari PM, Huang SM: Epidermal growth factor receptor modulation of radiation response: preclinical and clinical development. Semin Radiat Oncol 12:21-26, 2002 39. Nicholson RI, Hutcheson IR, Harper ME, et al: Modulation of epidermal growth factor receptor in endocrine-resistant, estrogen-receptor-positive breast cancer. Ann N Y Acad Sci 963:104-115, 2002 40. Wakeling AE, Nicholson RI, Gee JM: Prospects for combining hormonal and nonhormonal growth factor inhibition. Clin Cancer Res 7:4350s-4355s, 2001
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41. Gee JM, Hutcheson IR, Knowlden JM, et al: The EGFRselective tyrosine kinase inhibitor ZD1839 (Iressa) is an effective inhibitor of tamoxifen-resistant breast cancer growth. Proc Am Soc Clin Oncol 20:71a, 2001 (abstr 282) 42. Kokai Y, Myers JN, Wada T, et al: Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts. Cell 58:287-292, 1989 43. Muller W, Arteaga CL, Muthuswamy SK, et al: Synergistic interaction of the Neu proto-oncogene product and transforming growth factor alpha in the mammary epithelium of transgenic mice. Mol Cell Biol 16:5726-5736, 1996 44. Christensen JG, Schreck RE, Chan E, et al: High levels of HER-2 expression alter the ability of epidermal growth factor receptor (EGFR) family tyrosine kinase inhibitors to inhibit EGFR phosphorylation in vivo. Clin Cancer Res 7:4230-4238, 2001 45. Harris AL: Epidermal growth factor receptor in human breast cancer. Recent Results Cancer Res 113:70-77, 1989 46. 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 3:109-113, 1994 47. Agus DB, Akita RW, Fox WD, et al: Targeting ligandactivated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell 2:127-137, 2002 48. Totpal K, Lewis-Phillips GD, Balter I, et al: Augmentation of rhuMAb2C4 induced growth inhibition by Tarceva the EGFR tyrosine kinase inhibitor on human breast cancer cell line. Proc Am Assoc Cancer Res. 43:789, 2002 (abstr 3889) 49. Kerbel R, Folkman J: Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2:727-739, 2002 50. Ciardiello F, Caputo R, Bianco R, et al: Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clin Cancer Res 7:14591465, 2001 51. Petit AM, Rak J, Hung MC, et al: Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol 151:1523-1530, 1997
T
HE EPIDERMAL growth factor receptor (HER1/EGFR) is a member of the HER family of receptors. HER1/EGFR is a 170 kd transmembrane glycoprotein that has ligand-dependent intracellular tyrosine kinase (TK) activity. The ligands for HER1/EGFR, which include epidermal growth factor and transforming growth factor-␣ (TGF-␣), bind to HER1/EGFR with similar affinities.1 Ligand binding induces receptor phosphorylation and activates a specific set of cytoplasmic signaling molecules that constitute a complex signaling cascade.2 The mechanism of HER1/EGFR activation and signaling are not fully understood, but data suggest that overexpression and/or dysregulation of HER1/EGFR plays a pivotal role in tumor growth.3,4 Preclinical data show that HER1/ EGFR activation can promote tumorigenesis via a range of processes, including enhanced cellular proliferation, survival, migration, adhesion, and differentiation.2,5 Against this background, HER1/ EGFR was identified as an attractive target for the development of novel anticancer therapies. Several types of agents have been developed that Seminars in Oncology, Vol 30, No 3, Suppl 7 (June), 2003: pp 15-24
target HER1/EGFR. In particular, small-molecule TK inhibitors are recognized as an effective class of receptor antagonists with therapeutic potential. These agents inhibit TK activation and prevent the initiation of downstream signaling events. As a result, they block the cellular effects associated with HER1/EGFR activation.4 ERLOTINIB
Erlotinib HCl (Tarceva; Genentech Inc, South San Francisco, CA) is one of the most promising of a new generation of targeted therapeutics with potential efficacy against a broad range of solid tumors. It is an orally available, quinazoline-based, small-molecule HER1/EGFR-TK inhibitor.6 In vitro studies show that erlotinib inhibits purified HER1/EGFR with an IC50 of 2 nmol/L, while its potency for HER1/EGFR-TK in intact cells is about 10-fold lower.6 Importantly, erlotinib is highly selective for HER1/EGFR and shows minimal activity against other receptor and nonreceptor TKs in vitro. For example, erlotinib has an IC50 of 350 nmol/L for the HER2 TK and greater than 1,000 nmol/L for most other receptor TKs.6 Therefore, erlotinib can block the HER1/EGFR pathway without inhibiting other kinase-dependent pathways that may be essential for normal cellular function. This selectivity has important clinical ramifications for patient tolerability. The affinities of several TK inhibitors for HER1/EGFR and HER2 are shown in Table 1. Erlotinib and gefitinib (Iressa; AstraZeneca, Wilmington, DE), another HER1/EGFR-specific inhibitor, are both in advanced stages of clinical development for the treatment of a variety of tumors. Less specific TK inhibitors, for example, GW-2016 and PKI-166 that inhibit both HER1/EGFR and HER2 (dual inhibitors) and CI-1033 that inhibits all members
From the Department of Molecular Oncology, Genentech, Inc, South San Francisco, CA. Drs Akita and Sliwkowski are employees of Genentech Inc. Address reprint requests to Mark X. Sliwkowski, PhD, Department of Molecular Oncology, Genentech, Inc, South San Francisco, CA, 94080-4990. © 2003 Elsevier Inc. All rights reserved. 0093-7754/03/3003-0703$30.00/0 doi:10.1016/S0093-7754(03)00187-8 15
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Table 1. Affinity of Various Small-Molecule Tyrosine Kinase Inhibitors for HER1/EGFR and HER2
Compound
HER1/EGFR IC50 (mol/L)*
HER2 IC50 (mol/L)
Erlotinib Gefitinib GW-2016 PKI-166 CI-1033 EKB-509
0.02 0.023 0.01 0.025 0.0014 0.039 ⫾ 0.007
0.35 3.7† 0.009 NR 0.009 1.3 ⫾ 0.16
Abbreviations: IC50, 50% inhibitory concentration; NR, not reported. *Concentration required to inhibit 50% of HER1/EGFR in vitro. †Concentration required to inhibit 50% of the HER2 kinase in vitro. Source: Arteaga CL: The epidermal growth factor receptor: From mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol 19:32s-40s, 2001. Reprinted with permission of the American Society of Clinical Oncology.4
of the HER family (pan-HER inhibitor), are in preclinical or early phase clinical development. More data are required to assess the clinical potential of these dual and pan-TK inhibitors. Antitumor Activity Data from in vitro and in vivo preclinical studies show that erlotinib has substantial activity against a variety of tumor types. For example, in vitro studies using DiFi human colon tumor cells show that sub-micromolar concentrations of erlotinib inhibit cell-cycle progression and proliferation and initiate apoptosis.6 In murine xenograft models, orally administered erlotinib blocks ligand-induced HER1/EGFR autophosphorylation and substantially inhibits the growth of human head and neck tumor cells (HN5) with an ED50 of 9.2 mg/kg (Fig 1).7 In mice given a single 92 mg/kg oral dose of erlotinib, maximum HER1/EGFR inhibition was evident after 1 hour, HER1/EGFR autophosphorylation was reduced by 75% to 85% for at least 12 hours, and a significant reduction was observed 24 hours after treatment.7 Erlotinib also caused substantial inhibition of tumor growth in human non–small cell lung cancer (NSCLC) xenograft models developed using H460a or A549 cell lines.8 In the A549 model, tumor growth was
inhibited by 48% and 87% with erlotinib doses of 25 mg/kg and 100 mg/kg, respectively. These in vivo studies clearly show a direct relationship between HER1/EGFR inhibition and cessation of tumor growth. However, further preclinical studies are essential to enable us to fully understand how erlotinib-mediated inhibition of HER1/EGFR TK affects downstream signaling, and how these actions influence antitumor activity. This information should assist in optimizing the clinical application of erlotinib to maximize patient benefit. The effect of erlotinib on human pancreatic carcinoma xenografts implanted in immunodeficient mice has been studied by Ng et al.9 Tumors from two patients with pancreatic cancer were examined; one expressed high HER1/EGFR and moderate HER2 (OCIP#2) and the other expressed moderate HER1/EGFR and HER2 (OCIP#7). Erlotinib significantly inhibited HER1/ EGFR phosphorylation in both xenografts (Fig 2). However, the downstream effects on the phosphatidylinositol 3-kinase, protein kinase B (PI3KPKB/Akt) pathway and the ras-raf-mitogen–activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) pathway were more variable. Erlotinib significantly inhibited the levels of phosphorylated Erk in only one tumor (OCIP#2) and had no significant effect on PKB phosphorylation in either tumor.9 These differential effects may be the result of activation of these downstream effec-
Fig 1. Dose-dependent antitumor response with erlotinib against head and neck tumor xenografts in athymic mice. ED50, effective dose for 50% inhibition of the target receptor. (Modified and reprinted with permission of the American Society for Pharmacology and Experimental Therapies.7)
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Fig 2. Inhibition of HER1/ EGFR phosphorylation (pHER1/ EGFR) in human pancreatic orthotopic xenografts after erlotinib treatment. White bar, without erlotinib (control); striped bar, with erlotinib. (Modified and reprinted with permission of the American Association for Cancer Research.9)
tor molecules through alternative pathways, or possibly the incomplete inhibition of HER1/EGFR signaling by erlotinib. Nevertheless, these findings suggest that erlotinib may benefit patients with pancreatic cancer; a population with few, if any, treatment options beyond surgical intervention. A randomized, double-blind, placebo-controlled phase III clinical trial of erlotinib in combination with gemcitabine in patients with advanced pancreatic cancer (PA.3) is in progress. Erlotinib can also inhibit the growth of cells expressing the HER1/EGFR receptor mutant known as EGFRvIII (also referred to as delta EGFR or de2-7 EGFR). In contrast to wild-type HER1/EGFR, this deletion mutant lacks 267 residues in its extracellular domain, does not bind ligands, and has a constitutively active kinase.10-13 Expression of EGFRvIII has only been reported in tumors and is often associated with a more aggressive phenotype.14,15 In vitro studies show that EGFRvIII-expressing cells are sensitive to erlotinib, although to a lesser extent than cells with wild-type HER1/EGFR.16 These results are consistent with the observation that erlotinib is active against glioblastoma multiforme cells in vitro.17 HER1/EGFR gene amplification occurs in a high proportion of glioblastoma multiforme tumors, and the mutation that encodes EGFRvIII is frequently correlated with this amplification. These prelimi-
nary studies suggest that erlotinib may have utility against gliomas. Additional preclinical and phase I/II studies are in progress to evaluate the therapeutic potential of erlotinib in this indication. The Effect of Other HER Family Members on the Activity of Erlotinib Recent preclinical data show that erlotinib inhibits the growth of both high and modest HER1/ EGFR-expressing tumor cell lines. This observation suggests that HER1/EGFR expression is not the only prerequisite for the antitumor activity of erlotinib.8 Findings from clinical trials are consistent with this observation. They show that response to erlotinib does not appear to correlate with HER1/EGFR expression levels (data on file).18-22 However, unequivocal confirmation of this hypothesis is hindered by the lack of a reliable and reproducible method to quantify HER1/EGFR expression. HER2 is an important member of the HER receptor family. Although it has no known ligand, it potentiates cellular responses to ligand stimulation by forming heterodimers with HER1/EGFR and HER3, and altering their signaling characteristics. This occurs in several ways: association with HER2 increases the affinity of the receptors for their ligands; modulates their turnover rates; and provides a highly active kinase to the receptor
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Fig 3. Relative inhibition of HER1/EGFR and MAPK with erlotinib: MDA-MB-468 cells.24 F, HER1/EGFR; ■, MAPK.
complex.2 The latter effect is particularly significant in the case of HER3 because this receptor lacks intrinsic TK activity and is incapable of signaling in a homodimeric form. Moreover, HER2 is the preferred dimerization partner for HER1/EGFR and HER3.23 In vitro kinase assays show that erlotinib is 20- to 50-fold less effective against HER2 than HER1/EGFR.6 However, because HER2 modulates the signaling characteristics of HER1/EGFR, it is possible that the relatively weak inhibitory activity of erlotinib against the HER2 TK may have a disproportionately large effect on the overall signaling of HER1/HER2 heterodimers. This may, in part, explain the lack of correlation between HER1/EGFR expression and tumor responsiveness to erlotinib in clinical trials (data on file).18-22 In an effort to improve our understanding of the biological activity of erlotinib, dose-response experiments were performed using two HER1/EGFRoverexpressing cell lines (MDA-MB-468 and BT20) stimulated with TGF-␣, and two HER2/ HER3-expressing lines (MCF-7 and MDA-MB453) stimulated with heregulin.24 The results of this study indicate that although increasing doses of erlotinib correlate positively with inhibition of receptor activation and downstream signaling, these effects are not directly proportional.24 For example, the dose–response curves indicate that although 90% of the TGF-␣–stimulated HER1/ EGFR activity in MDA-MB-468 cells is inhibited by 540 nmol/L erlotinib, only 33% of the MAPK activity is blocked at that concentration (Fig 3).
MDA-MB-468 cells express approximately 2 ⫻ 106 HER1/EGFRs per cell.25 Therefore, the 200,000 HER1/EGFRs still capable of signaling after treatment with 540 nmol/L erlotinib can stimulate 67% of the maximum MAPK activity. These data imply that activation of only a small fraction of the HER1/EGFRs is sufficient to generate a substantial mitogenic signal in cells that highly overexpress the receptor. Effective inhibition of signaling by HER1/EGFR-targeted agents may only occur at very high levels of receptor inhibition. Therefore, it is reasonable to propose that HER1/EGFR-targeted agents should be used at doses that are as high as possible (ie, the maximum tolerated dose) to give the best chance of effectively inhibiting not only receptor activation, but also downstream signaling. Erlotinib-mediated inhibition of downstream signaling and receptor phosphorylation are also disproportionate in HER2-expressing cells; however, the relationship is quite different. The same 540 nmol/L concentration of erlotinib inhibits only 28% of the heregulin-stimulated HER2 activity in MCF-7 cells, but blocks almost 90% of the MAPK activity (Fig 4). The extent to which erlotinib inhibits receptor phosphorylation in heregulin-stimulated HER2/HER3-expressing cells was approximately 50-fold lower than for TGF-␣– stimulated receptor phosphorylation in HER1/ EGFR-expressing cells.24 However, the IC50 for erlotinib-mediated inhibition of p42 and p44 MAPK phosphorylation was within a similar range in both cases: approximately 200 nmol/L for
PRECLINICAL STUDIES WITH ERLOTINIB
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Fig 4. Relative inhibition of HER2 and MAPK with erlotinib: MCF-7 cells.24 F, HER2/3; ■, MAPK.
heregulin-stimulated HER2/HER3-expressing cells and 500 to 700 nmol/L for TGF-␣–stimulated HER1/EGFR expressing cells. In summary, these findings show that, despite the much lower potency of erlotinib against the HER2 kinase in vitro, it blocks ligand-mediated signaling of HER1/ EGFR and HER2/HER3 with similar potencies. These in vitro results suggest that erlotinib may also be effective against tumors that are dependent on HER2 activation for growth and/or survival. This may increase the range of potential patients that could benefit from treatment with this inhibitor. Studies examining the effects of other HER1/ EGFR inhibitors on tumors that coexpress HER1/ EGFR and HER2 show findings similar to those with erlotinib. In a recent report, treatment with gefitinib resulted in a significant dose-dependent reduction in the level of HER1/EGFR and HER2 tyrosine phosphorylation. Phosphorylation of MAPK and Akt were also reduced.26 In another study, Moulder et al27 found that treatment with 1 mol/L gefitinib substantially inhibited HER2-TK phosphorylation. Finally, AG1478, another quinazoline derivative that inhibits HER1/ EGFR, suppressed the growth of mammary tumors and blocked HER2 phosphorylation in MMTV/ TGF ⫻ MMTV/neu (the mouse homologue of human HER2) bigenic mice.28 Taken together, these data suggest that HER1/EGFR inhibitors could be used to treat patients with solid tumors that express HER1/EGFR and HER2. HER3 and HER4 are also involved in ligand-induced het-
erodimerizations with HER1/EGFR, but the effects of erlotinib on the activation and signaling of these receptor combinations has not been examined in detail. ERLOTINIB IN COMBINATION WITH CONVENTIONAL ANTICANCER THERAPIES
There is evidence to suggest that HER1/EGFRdriven tumor cells, particularly those in which autocrine activation may play a role, can become resistant to chemotherapeutic agents or antihormonal therapy.29 In breast and prostate tumors, the development of an autocrine loop is thought to be a pivotal step in the progression to a hormone-independent state.30,31 Also, a substantial amount of preclinical data from studies with various agents shows that blocking HER1/EGFR activity inhibits the ability of tumor cells to repair the damage caused by chemotherapy or radiation.4,29,32 Therefore, HER1/EGFR overexpression and/or dysregulation may affect the success of many conventional cancer therapies. It is common clinical practice to combine various chemotherapeutic agents and radiotherapy because this approach often improves efficacy and decreases the chance of resistance.33 Against this background, there is a strong rationale to examine the effectiveness of combining erlotinib with chemotherapeutic agents, radiotherapy, and/or antihormonal therapy.
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Table 2. Erlotinib in Combination With Various Chemotherapeutic Agents Combinations With Erlotinib Agent Cisplatin Doxorubicin HCl 5-fluorouracil Paclitaxel Vinorelbine tartrate Gemcitabine HCl
Dose/Route/Regimen 10 mg/kg IV daily ⫻ 1 15 mg/kg IV daily ⫻ 1 200 mg/kg IP daily ⫻ 1 10 mg/kg IP daily ⫻ 5 20 mg/kg IP daily ⫻ 5 25 mg/kg IV daily ⫻ 3 100 mg/kg IP three times a day ⫻ 4
Lethality No No No No No No No
increase increase increase increase increase increase increase
Efficacy Additive effects, no antagonism Additive effects, no antagonism No interaction, no anatagonism Additive effects, no anatagonism No interaction, no antagonism No interaction, no antagonism Additive effects, no antagonism
Abbreviations: IV, intravenous; IP, intraperitoneal. Data on file, Genentech, Inc and OSI Pharmaceuticals, Inc, 2002.
Combination With Chemotherapy The effect of combining erlotinib with several commonly used chemotherapeutic agents was examined in human tumor xenograft models. The results are summarized in Table 2. In all studies, erlotinib was administered at the ED50 (9.2 mg/kg/ day).7 No antagonism of the therapeutic efficacy of any chemotherapeutic agent was observed. Erlotinib in combination with cisplatin, doxorubicin, or gemcitabine had an additive effect on antitumor activity. Additive effects were also observed with paclitaxel at 10 mg/kg, but not 20 mg/kg (data on file).7 Importantly, the toxicities of these chemotherapeutic agents, and the toxicity of erlotinib itself, were not exacerbated in any combination. These data strongly suggest that using erlotinib in combination with certain chemotherapeutic agents may substantially enhance antitumor activity without adversely affecting toxicity. Consequently, clinical trials have been initiated to examine the feasibility and efficacy of several of these combinations. The findings from these preclinical studies also show that the overall antitumor activity achieved when agents are combined is not dependent on the dosing sequence (Fig 5).7 There is no clear evidence to suggest that the sequence of administration is important when erlotinib is combined with these chemotherapeutic agents. However, this may not be the case for all combinations and indications. Additional preclinical studies are in progress to elucidate the mechanism of these additive effects. Phase Ib trials examining erlotinib in combina-
tion with temozolomide; docetaxel; capecitabine; gemcitabine; gemcitabine and cisplatin; carboplatin and paclitaxel; capecitabine and docetaxel; 5-fluorouracil, leucovorin, and oxaliplatin; and docetaxel and carboplatin are currently underway in patients with various advanced solid tumors. Preliminary data from studies of erlotinib in combination with docetaxel,34 gemcitabine and cisplatin,35 and carboplatin and paclitaxel36 are encouraging, showing evidence of antitumor activity and no significant adverse pharmacokinetic interactions between the agents. Importantly, two randomized, double-blind, placebo-controlled phase III clinical trials, TRIBUTE and TALENT, are in progress examining erlotinib in combination with standard chemotherapy as first-line therapy for patients with advanced NSCLC. Combination With Radiotherapy Preclinical data have shown that erlotinib can enhance the effects of radiation in H460 NSCLC cells and H460 NSCLC xenografts implanted in mice.37 The precise mechanism that causes this sensitization to radiation is unknown, but it is proposed that a combination of HER1/EGFR inhibition and downregulation of COX-2 expression are involved. Based on these data, trials of erlotinib in combination with radiotherapy are planned. Several other studies show that other HER1/EGFR inhibitors can also enhance the antiproliferative effects of radiation in vitro and in vivo.38
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Fig 5. Erlotinib in combination with cisplatin: The effect of scheduling. i.v., intravenous; i.p., intraperitoneal; q.d./q.i.d., once daily; t.i.d., three times daily. (Modified and reprinted with permission of the American Society for Pharmacology and Experimental Therapies.7)
Combination With Antiestrogen Therapy There is increasing evidence to suggest that HER1/EGFR signaling pathways are critical for the hormone independence of estrogen receptorpositive breast cancers.39 For example, in vitro studies show that resistance to tamoxifen is associated with upregulation of HER1/EGFR and its signaling pathway.40 Consequently, HER1/EGFR inhibitors could potentially be used to treat antihormone-resistant breast tumors. Preclinical studies show that erlotinib combined with tamoxifen has an additive effect against estrogen receptorpositive HER2-overexpressing human breast cancer cell lines (MDA-361 and MCF-7/HER2; C. Arteaga, personal communication, September 2002). Also, Gee et al41 showed that gefitinib effectively inhibits tamoxifen-resistant breast cancer cell growth in vitro. In view of these data, phase II trials examining erlotinib in combination with letrozole (Femara; Novartis, Basel, Switzerland), a reversible, nonsteroidal aromatase inhibitor, are planned. ERLOTINIB IN COMBINATION WITH TARGETED AGENTS
The regulation of tumor growth and progression is complex, involving many receptors, intracellular
pathways, and genes. Inhibiting more than one crucial pathway or process could be an effective clinical strategy. The advent of novel targeted agents that inhibit receptors and pathways involved in tumorigenesis offer many possibilities for combining various agents with the aim of improving overall therapeutic efficacy. Preclinical studies have a vital role in identifying the most effective combinations before designing clinical trials. These studies are still at an early stage. HER2 Inhibitors Because HER2 is a critical element in the HER signaling network, inhibiting HER2 as well as HER1/EGFR is an attractive proposition. Studies on transfected fibroblasts and transgenic mice show that the growth of tumors induced by excessive HER1/EGFR signaling can be accelerated by the concomitant overexpression of neu (the mouse homologue of HER2).42,43 In vivo studies show that a higher concentration of TK inhibitor is required to inhibit HER1/EGFR phosphorylation in tumors that express both HER1/EGFR and HER2, as opposed to HER1/EGFR alone.44 Moreover, clinical studies in breast and NSCLC show that overexpression of both receptors correlates
22
with poorer overall survival compared with overexpression of HER1/EGFR alone.45,46 Preclinical studies of various HER1/EGFR inhibitors and trastuzumab (Herceptin; Genentech, Inc), an anti-HER2 humanized monoclonal antibody, show that HER1/EGFR inhibitors can interfere with HER1/HER2 crosstalk and downregulate HER2 activity, although the exact mechanisms are not fully understood. For example, the addition of gefitinib and trastuzumab to the breast cancer cell line SK-BR-3 resulted in a greater inhibition of cell growth than with either agent alone.26 Preliminary evidence suggests a similar outcome when erlotinib is combined with trastuzumab (C. Arteaga, personal communication, September 2002). Therefore, combining a HER1/EGFR inhibitor, such as erlotinib, with trastuzumab in the clinical setting could have a synergistic inhibitory effect on tumorigenesis, resulting in improved antitumor effects. 2C4 (pertuzumab) is a humanized monoclonal antibody that blocks the association of HER2 with other HER receptors and prevents ligand-dependent and ligand-independent HER2 signaling.47 This is in contrast to trastuzumab, which acts only against the ligand-independent receptor activation associated with HER2 overexpressing tumors. Thus, 2C4 may have activity in patients that would not be candidates for trastuzumab treatment. Its unique mechanism of action suggests that 2C4 has the potential to be highly effective alone and in combination with HER1/EGFR-targeted agents. The effects of combinations of 2C4 and erlotinib on the growth of MDA-MB-175 breast tumor cell lines were examined in a recent study.48 Suboptimal concentrations of both drugs used together resulted in greater caspase 3/7 activation than the same concentration of either agent alone. These data suggest that combined treatment with erlotinib and 2C4 may be efficacious against tumors that do not respond well to single-agent erlotinib. Phase I and II clinical trials of 2C4 monotherapy in various tumors are in progress. Vascular Endothelial Growth Factor-Targeted Agents The HER1/EGFR pathway is involved, to a certain extent, in the production of angiogenic factors such as vascular endothelial growth factor (VEGF).49 Several studies show that HER1/ EGFR-targeted agents can downregulate VEGF
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production and angiogenesis in human cancer cells.50,51 Combining HER1/EGFR inhibitors with VEGF/angiogenesis inhibitors is an attractive strategy. The potentially complimentary mechanisms of action of these inhibitors may have additive effects on angiogenesis and other VEGF-related actions. These effects could result in a substantial inhibition of tumor growth. To this end, a phase I clinical trial of erlotinib in combination with bevacizumab (Avastin; Genentech Inc), an anti-VEGF monoclonal antibody, for advanced, refractory NSCLC is in progress. CONCLUSION
The biology and genetics underlying the development and progression of solid tumors are complex and involve many different signal transduction pathways. Over the past two decades, however, it has become clear that members of the HER family of growth factor receptors play central roles in the initiation or maintenance of a number of different cancers. Consequently, these receptors have become attractive therapeutic targets. Several agents that specifically target either the HER1/EGFR or HER2 receptors have recently been developed. Findings thus far support the use of these agents in combination with traditional chemotherapy, radiotherapy, and targeted drugs. These new agents are currently being evaluated in combination with existing therapeutic regimens in the treatment of patients with advanced refractory disease. Interpretation of the results from these clinical trials is complicated by the fact that the efficacy of these drugs often does not correlate with the level of expression of their target receptors. This is likely a reflection of the complexity of the signaling network and the fact that the signaling of one HER family member can be modified by heterodimerization with a different member. Preclinical studies are vital for identifying the most important signaling pathways and for elucidating how the interactions of the various HER receptors affect these pathways. These studies promise to improve the probability of positive clinical trial outcomes by allowing for more rational, signaling pathway-based trial designs. This review highlights findings from recent preclinical studies that provide a new perspective on the relationship between erlotinib and the HER signaling network. More studies to examine the actions and interactions of HER1/EGFR and other
PRECLINICAL STUDIES WITH ERLOTINIB
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