Development of strategies for the use of anti-growth factor treatments
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《肿瘤与内分泌》
1 Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Cardiff, UK
2 Dip. Medicina Sperimentale e Diagnostica, Universita di Ferrara, Ferrara, Italy
This paper was presented at the 1st Tenovus/AstraZeneca Workshop, Cardiff (2005). AstraZeneca has supported the publication of these proceedings.
Abstract
Aberrant signalling through the epidermal growth factor receptor (EGFR) is associated with increased cancer cell proliferation, reduced apoptosis, invasion and angiogenesis. Over-expression of the EGFR is seen in a variety of tumours and is a rational target for antitumour strategies. Among the classes of agent targeting the EGFR are small-molecule inhibitors, which include gefitinib (IRESSATM), which acts by preventing EGFR phosphorylation and downstream signal transduction. De novo and acquired resistance, however, have been reported to gefitinib and here we describe evidence which indicates that the type II receptor tyrosine kinases (RTKs) insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (InsR) play important roles in the mediation of responses to gefitinib in the de novo- and acquired-resistance phenotypes in several cancer types. Moreover, combination strategies that additionally target the IGF-IR/InsR can enhance the antitumour effects of gefitinib.
Introduction
Advances in the understanding of the aberrant signalling pathways involved in the development and progression of cancer have resulted in the development of therapeutic agents or so-called smart drugs that target specific components of these pathways, the aim being that such precise targeting would hopefully produce greater activity and specificity against the tumour cell than is currently observed with traditional chemotherapeutic drugs. Based on these criteria, among the first and extremely successful targeted therapies in oncology could considered to be the development of anti-hormone therapies for breast and prostate cancer which blocked the action of the oestrogen receptor (ER) and androgen receptor respectively, with the majority of patients with hormone-responsive tumours showing good responses to this form of treatment (Griffiths et al. 1997, Gee et al. 2002). Over the last 20 years, advances in molecular diagnostics, complemented by the advent of high-throughput screening of potential anti-cancer compounds, has resulted in the development of novel drugs that can focus on targets with a specificity hitherto unavailable. This has been particularly evident in the emergence of inhibitors of specific signal transduction kinases, enzymes with critical roles in the pathways involved in the transfer of external stimuli such as hormones and growth factors to the nucleus. Thus, from this new genre of targeted anticancer therapies, notable successes include the humanized monoclonal antibody trastuzumab (HerceptinTM) for HER-2/neu-over-expressing metastatic breast cancer; the treatment of bcr/abl-translocation-positive chronic myelogenous leukaemia with the tyrosine kinase inhibitor (TKI) imatinib (GleevecTM); the use of cetuximab (ErbituxTM) in colorectal cancer; a chimeric monoclonal antibody that binds to the epidermal growth factor (EGF) receptor (EGFR); bevacizumab (AvastinTM), a humanized murine monoclonal antibody targeting the vascular endothelial growth factor and, finally, in the treatment of non-small cell lung carcinoma (NSCLC) the utilization of gefitinib and erlotinib (TarcevaTM), which are small-molecule EGFR TKIs (reviewed by Ross et al. 2004).
It is apparent, however, that despite the valuable role that these novel drugs may have in the treatment of malignancies, providing a much-needed therapeutic option for patients with advanced disease and often extremely poor prognosis, the clinical results overall have not equated with the expectations of such exciting specific therapies. Consequently, this article will focus on the smart strategies used to target a key player implicated in cancer development and disease progression, namely the EGFR, and discuss not only the successes but also the failures of these therapies, with particular focus on gefitinib in breast cancer. Furthermore, utilizing the current literature and work undertaken in our laboratory, we will describe mechanisms that may relate to a lack of response to gefitinib and postulate ways in which the efficacy and duration of response may be improved.
EGFR signalling
The EGFR belongs to the erbB receptor family which consist of four members: erbB1/HER-1/EGFR, erbB2/HER-2/neu, erbB3 and erbB4. The EGFR is a membrane glycoprotein that consists of a ligand-binding region, a transmembrane segment and an intracellular portion, the latter containing a tyrosine kinase domain upstream of additional autophosphorylation tyrosine sites within the C-terminus of the receptor (Gullick 2001). Activation of the EGFR occurs when ligands such as EGF, transforming growth factor- (TGF-) or amphiregulin bind to the EGFR causing receptor dimerization, either with another EGFR monomer or with another member of the erbB family. Dimerization subsequently activates the tyrosine kinase domains of each receptor resulting in the trans-autophosphorylation of the tyrosine sites on the other receptor molecule. These phosphorylated residues ultimately act as binding sites for adaptor proteins which recruit and activate a variety of signalling transduction cascades involved in cell proliferation and survival (Gullick 2001). Important pathways in erbB receptor signalling are the Ras/Raf/mitogen-activated protein kinase (MAPK) cascade, which is usually associated with cell proliferation, and the phosphoinositide 3-kinase (PI 3-kinase)/Akt route, which mediates numerous biological processes such as cell survival, gene expression and cell-cycle progression.
EGFR in cancer and EGFR-targeted strategies
The EGFR is expressed in a wide range of solid tumours including breast and prostate and has been implicated as playing a major role in tumour growth and progression (Salomon et al. 1995, Nicholson et al. 2001a). Moreover, many solid tumours also demonstrate elevated expression of the cognate ligands for EGFR, namely EGF and TGF- (Salomon et al. 1995). It has also been shown in breast cancer that lack of response to endocrine therapy, together with poor survival and increased metastasis, is associated with over-expression of EGFR (Nicholson et al. 1994, 2001b). Additionally, in vitro studies have demonstrated that transfection of EGFR into hormone-dependent breast tumour cells can mediate acquired resistance to the anti-oestrogen therapy tamoxifen (van Agthoven et al. 1992), such resistance being a major problem in the treatment of clinical breast cancer (Gee et al. 2002). Furthermore, we have demonstrated in our laboratory that acquired resistance to the pure anti-oestrogen fulvestrant (FaslodexTM) and tamoxifen (NolvadexTM) can be facilitated by EGFR-mediated growth pathways. These resistant cell lines show increased EGFR expression/signalling and are highly sensitive to the EGFR TKI gefitinib (McClelland et al. 2001, Knowlden et al. 2003). Similarly to breast cancer, aberrant EGFR signalling has been linked to the progression of hormone-responsive prostate cancer to the hormonerefractory state (Djakiew 2000).
Clearly, the EGFR represents an important target in cancer therapy and this has prompted the development of a variety of different agents to prevent EGFR signal transduction. These agents have been reviewed elsewhere (Ciardiello & Tortora 2001) but briefly, the most advanced in clinical development include small-molecule TKIs such as gefitinib and erlotinib, both of which are quinazolines and act by competitively inhibiting ATP binding to the tyrosine kinase domain of the EGFR, thus preventing EGFR autophosphorylation and subsequent downstream signal transduction, and the monoclonal antibodies cetuximab and ABX-EGF, which bind to the external domain of the EGFR. Indeed, both gefitinib and erlotinib have now been approved for the treatment of advanced NSCLC and cetuximab has been approved for advanced colorectal cancer.
Gefitinib: preclinical and clinical evaluation
Preclinical studies have indicated that gefitinib is growth-inhibitory to a range of human tumour xenografts and, importantly, potentiates the activity of various cytotoxic agents when used in combination studies (Ciardiello et al. 2000, Wakeling et al. 2002). Evidence shows that gefitinib was growth-inhibitory in xenografts of both ER-positive and ER-negative ductal carcinoma in situ tissue (Chan et al. 2002) and, furthermore, the inhibitor could restore tamoxifen sensitivity in de novo resistant HER-2-overexpressing MCF-7 human breast cancer xenografts (Massarweh et al. 2002). Gefitinib was extremely effective at preventing EGFR autophosphorylation and the subsequent downstream activation of extracellular- signal-regulated kinase (ERK) 1/2 MAPK and Akt in our breast cancer models of tamoxifen or fulvestrant resistance, under both basal and EGF-ligand- primed conditions (McClelland et al. 2001, Nicholson et al. 2001b, 2004, Knowlden et al. 2003). In addition, cell growth of each model was inhibited dramatically (90%), which was in marked contrast to the minimal effect (<10%) that the inhibitor displayed on the growth of the parental MCF-7 cells (McClelland et al. 2001, Nicholson et al. 2001b, 2004, Knowlden et al. 2003). Overall, such data indicate that gefitinib may be valuable in the treatment of EGFR expressing ER-negative and ER-positive breast tumours.
The clear rationale for the use of gefitinib in breast cancer has given rise to numerous clinical trials with the inhibitor in this cancer type. Indeed, gefitinib is currently undergoing evaluation in breast cancer in a range of Phase II studies. Excitingly, we have shown, in a Phase II study, that gefitinib produced worthwhile clinical benefit in ER-positive patients with acquired tamoxifen resistance, with 11 out of 13 patients demonstrating partial responses and disease stabilization, as well as an extended median time to progression (Gee et al. 2004, Gutteridge et al. 2004). Additionally, another Phase II trial assessing the efficacy of gefitinib monotherapy in patients with advanced breast cancer showed that 10 out of 33 patients demonstrated stable disease (Baselga et al. 2003).
EGFR status and response to gefitinib
It has become apparent however that although clinical benefit has been observed with gefitinib in numerous solid tumour types reported to over-express EGFR including breast, the clinical data have also disappointingly revealed the existence of de novo and acquired resistance to gefitinib therapy with some patients deriving no clinical benefit or displaying varying durations of response prior to disease relapse (Baselga et al. 2002, Ranson 2002, Ranson et al. 2002, Kelly & Averbuch 2004). Critically, it has now been established that although there is a strong correlation between EGFR levels and aggressive disease with reduced survival (Nicholson et al. 2001b), a clear association between EGFR expression/phosphorylation and predicted response to various EGFR-targeted agents including gefitinib does not exist (reviewed by Arteaga 2002). Moreover, in our clinical study with gefitinib, in advanced breast cancer patients who were either ER-positive with acquired tamoxifen resistance or who had de novo hormone-resistant ER-negative disease, the gefitinib-responsive patients were in fact the former group who expressed only modest EGFR prior to and after 8 weeks gefitinib treatment. Surprisingly, 85% of the ER-negative/over-expressing EGFR patients had de novo resistance to gefitinib (Gee et al. 2004). Interestingly, this equates with our in vitro model system data for ER-positive acquired tamoxifen resistance, where we have shown conclusively that modestly increased EGFR signalling is growth-contributory (Knowlden et al. 2003). A proportion of these responding patients (five out of nine) also showed an obvious fall in phosphorylated EGFR and activated MAPK (Gee et al. 2004); however, decreases in EGFR phosphorylation were not observed in all patients showing clinical benefit (Gee et al. 2004), suggesting that a non-classical gefitinib-response mechanism may operate in some patients.
Such observations have been noted in other cancer types. For example, EGFR is over-expressed in up to 70% of NSCLC (Rusch et al. 1997, Fontanini et al. 1998) yet only modest objective responses of between 12 and 18% were observed with gefitinib in Phase II trials in this cancer type (Fukuoka et al. 2003, Kris et al. 2003). Moreover, from these trials, it has been determined that EGFR membrane staining predicts neither tumour response nor symptom improvement in NSCLC (Bailey et al. 2003). Interestingly however, recent research has identified specific somatic mutations in the EGFR tyrosine kinase domain in a subset of NSCLC patients who showed a dramatic improvement from gefitinib (Lynch et al. 2004, Paez et al. 2004), which may aid patient selection. It has also been observed that responses to another anti- EGFR agent, namely erlotinib, in patients with NSCLC did not show any correlation to EGFR staining (Perez-Soler et al. 2004). It is therefore abundantly clear that considerable research is required to not only define the mechanisms which promote both acquired and de novo resistance to EGFR inhibitors with the aim of improving efficacy and duration of response in a greater number of patients but additionally, delineate other molecular markers which may predict sensitivity to EGFR inhibitors such as gefitinib.
Characteristics of acquired resistance to gefitinib
Up-regulation of the type II receptor tyrosine kinase (RTK) insulin-like growth factor-I receptor (IGF-IR) signalling
The varying durations of response (3–12 months) displayed by responders to gefitinib in various different cancer types (Barton et al. 2001, Baselga et al. 2002, Ranson 2002, Ranson et al. 2002, Douglass 2003, Schiller 2003, Kelly & Averbuch 2004) indicated the fairly rapid acquisition of resistance to the TKI. In our laboratory, we have generated several model systems in breast, prostate and lung cancer to study this phenomenon in vitro. For example, utilizing our in vitro model system for acquired tamoxifen resistance, which consists of EGFR-positive, MCF-7- derived, tamoxifen-resistant breast cancer cells (Knowlden et al. 2003), we continuously exposed this cell line, designated TAM-R, to 1 μM gefitinib, a concentration shown to be growth-inhibitory to the TAM-R cells (Knowlden et al. 2003). A sustained growth inhibition (90%) was observed for 4 months before the surviving cells resumed proliferation and a stable gefitinib-resistant subline (TAM/TKI-R) was established after a further 2 months, which showed no detectable basal phosphorylated EGFR activity and minimal MAPK activity (Jones et al. 2004a). Compared with the parental TAM-R cells, however, the TAM/TKI-R cells demonstrated elevated levels of phosphorylated IGF-IR and, furthermore, an increased sensitivity to growth inhibition by the IGF-IR TKI, AG1024 (Calbiochem, Merck Biosciences, Nottingham, UK; Jones et al. 2004a).
Over-expression and activation of the IGF-IR, a transmembrane tyrosine kinase receptor, and its downstream signalling molecules have been linked to disease progression in breast cancer and other cancer types (Yu & Rohan 2000). In addition, the TAM/TKI-R cell line also showed elevated levels of activated Akt and protein kinase C (PKC) , which could be modulated by treatment with AG1024, suggesting that these components were downstream targets for IGF-IR signalling in these cells (Jones et al. 2004a). Moreover, preliminary data from another of our MCF-7-derived breast cancer cell lines dually resistant to the pure-anti-oestrogen faslodex and gefitinib (FAS/TKI-R) indicates that compared with their faslodex-resistant parents (FASR) (McClelland et al. 2001), the FAS/TKI-R cells also demonstrate elevated expression and activation of IGF-IR and PKC (HE Jones, JMW Gee, D Barrow, M Rubin & RI Nicholson; unpublished observation), the significance of which is currently under investigation.
Overall, such findings indicate that IGF-IR is an important therapeutic target in acquired gefitinib resistance in breast cancer and strategies which target this receptor may increase the efficacy and duration of response to gefitinib. A crucial question is, however, whether IGF-IR signalling is important in mediating gefitinib responses in other cancer types. To this end, we have shown additionally that androgen-independent prostate cancer cells with acquired gefitinib resistance also show a dramatic marked increase in the expression and activation of membrane-associated IGF-IR, together with considerable increased levels of the ligand IGF-II and, furthermore, a dependence on IGF-IR for growth (Jones et al. 2004a), thus supporting the importance of IGF-IR signalling in the acquired-gefitinib-resistance phenotype. Indeed, evidence is accumulating which indicates that an association exists between elevated IGF-IR expression/signalling and its downstream components such as Akt and resistance to drugs which inhibit erb-family signal transduction in various cancers. For example, IGF-IR via PI 3-kinase/Akt activation has been shown to mediate resistance to the anti-EGFR monoclonal antibody 225 in glioblastoma cells (Chakravati et al. 2002) and also the selective EGFR TKI AG1478 (Sigma Chemical Co., St Louis, MO, USA) in the DiFi human colorectal cancer cell line (Liu et al. 2001).
Elevated HER-2 (erbB-2, neu) signalling
It is established that the erbB receptor HER-2 is amplified and over-expressed in approximately 30% of breast cancers and that, furthermore, such overexpression is associated with aggressive metastatic disease with reduced time to relapse (Slamon et al. 1987). A ligand for HER-2 has not been clearly defined; however, through heterodimerization HER-2 can mediate the signal transduction of all other erbB family members when they bind their cognate ligands (Gullick 2001). Interestingly, we have shown that in our tamoxifen-resistant breast cancer cells the expression of both EGFR and HER-2 is increased but, moreover, HER-2 preferentially dimerizes with EGFR in these cells which subsequently show good responses to gefitinib (Knowlden et al. 2003) and the HER-2 inhibitor trastuzumab (Fig. 1A). Once these cells acquire resistance to gefitinib, however, levels of HER- 2 expression and activity are further increased (Fig. 1B) but, paradoxically, sensitivity to trastuzumab is lost (Fig. 1A). Thus, gefitinib resistance has generated cross-resistance to another agent, namely trastuzumab. Interestingly, IGF-IR signalling has also been implicated in modulating the responses to trastuzumab (Lu et al. 2001). In addition, it has also been observed that IGF-IR can unidirectionally activate HER-2, which involves a physical association of the two receptors (Balana et al. 2001). Our gefitinib-resistant breast cancer cells also show evidence of the existence of a physical interaction between the IGF-IR and HER-2, which furthermore can co-localize in areas at the tumour cell membranes (Fig. 2A and B). Additionally, preliminary work excitingly shows that concentrations of the IGF-IR TKI AG1024 that block IGF-IR phosphorylation also inhibited HER-2 activity (Fig. 2C). Interestingly, other workers have shown that the IGF-IR-mediated attenuation of trastuzumab growth inhibition is dependent upon signalling via PI 3-kinase/Akt and not MAPK in HER-2 over-expressing breast cells (Lu et al. 2004). Similarly, we have also shown that in our gefitinib-resistant breast cancer cells, which have elevated HER-2 and IGF-IR activity, Akt phosphorylation is increased and that IGF-IR signalling appears to be dissociated from MAPK (Jones et al. 2004a). Such data adds further to the observation that the IGF-IR is increasingly becoming a key therapeutic target to potentially improve the efficacy of erbB inhibitors.
Maintenance of ER phosphorylation in acquired gefitinib-resistant breast cancer
In our TAM/TKI-R acquired-resistance gefitinib model, ER expression is retained at levels comparable to the parental TAM-R and MCF-7 cells and, furthermore, this ER appears functional, since the anti-oestrogen fulvestrant decreases the growth of the TAM/TK1-R cells and these cells express increased oestrogen-regulated gene levels (HE Jones, M Giles, JMW Gee, D Barrow, AE Wakeling & RI Nicholson; unpublished observation). Interestingly, the ER appears to be highly phosphorylated at serine-118 in such cells, its activity even exceeding the high level observed in their TAM-R parents (Britton et al. 2002). Moreover, this occurs despite lower levels of activated MAPK, the kinase implicated in phosphorylation of this site in the parental TAM-R cells (Britton et al. 2003). Intriguingly, we have shown using immunopreciptation and immunofluorescence that activated PKC, significantly elevated in TAM/TKI-R and a downstream target for IGF-IR (Jones et al. 2004a), can physically interact with and co-localize with activated nuclear ER. Moreover, the PKC inhibitor bisindoylmaleimide IX (Ro-31-8220; Calbiochem) blocks serine-118 ER phosphorylation and reduces expression of ER-regulated genes, suggesting productive crosstalk between ER and PKC in these gefitinib resistant cells (HE Jones, M Giles, JMW Gee, D Barrow, AE Wakeling & RI Nicholson; unpublished observation). In summary, these data suggest that increased IGF-IR-driven, nuclear-activated PKC may contribute to growth of acquired gefitinib-resistant breast cancer cells via its recruitment to and phosphorylation of ER to promote cell growth. Interestingly, our additional acquired gefitinib-resistant breast cancer model, derived from FASR breast cancer cells, shows very low levels of ER but is still phosphorylated and is coupled with increased expression of oestrogen-regulated genes compared with the parental cells. Since these cells also show increased IGF-IR/nuclear PKC signalling versus their parental cells, it is possible that the PKC/ER crosstalk mechanism may again be contributory.
Importance of type II RTKs in partial or de novo gefitinib resistance
It is known that EGFR signalling can be modulated by several mechanisms which include heterodimerization with other members of the EGFR family, such as HER-2 (erbB-2; Gullick 2001), and transactivation by, or crosstalk with, other growth factor receptors such as IGF-IR (Roudabush et al. 2000, Wang et al. 2002) or steroid hormone receptors (Lichtner 2003). Indeed, we have shown that in our tamoxifen-resistant breast cancer model the IGF-IR appears to be permissive for EGFR signalling (Nicholson et al. 2004). Importantly, we have demonstrated that similarly to acquired gefitinib resistance in breast and prostate cancer, the efficacy of gefitinib may be compromised in de novo resistance in colorectal cancer cells by the involvement of the type II RTK insulin receptor (InsR) isoform-A (InsR-A; Jones et al. 2004b), a closely related family member to the IGF-IR and which has been linked to cancer development and disease progression (reviewed by Denley et al. 2003). Indeed, data indicated that, via EGFR blockade, gefitinib can facilitate the activity of the InsR which in turn can modulate EGFR phosphorylation (Jones et al. 2004b). Excitingly, preliminary work in our laboratory also suggests a major role for the involvement of the IGF-IR in the partial resistance to gefitinib displayed by NSCLC cells. It is also feasible that in tumour cells that, are insensitive or only partially responsive to gefitinib, existing high IGF-IR/InsR activity may also play an important role in the maintenance of cell proliferation independently of the EGFR.
Strategies to improve gefitinib response with other signal transduction inhibitors
Targeting the IGF-IR
Following the elucidation of the mechanisms underlying the acquired and de novo resistance to gefitinib, combination strategies involving other signal transduction inhibitors which inhibit these resistance mechanisms would hopefully extend durations of response and improve the efficacy of gefitinib. It is clear that the type II RTKs, in particular the IGF-IR, represent important targets in the search to improve gefitinib response not only in breast and prostate cancer but also in other cancer types. Indeed, numerous pharmaceutical programmes have been implemented to develop specific inhibitors of the IGF-IR but this is still in the early stages of discovery; however, potential anti-IGF-IR strategies have been reviewed by Surmacz (2003). We have already shown that the acquisition of gefitinib resistance in our tamoxifen-resistant breast cancer cells can be delayed or even prevented by the combination of gefitinib plus an IGF-IR inhibitor (Nicholson et al. 2004) and, excitingly, early studies indicate that in the presence of an IGF-IR inhibitor gefitinib effects can be observed on tumour cells that had previously demonstrated de novo resistance to this inhibitor.
Targeting other elements in combination with gefitinib
There is considerable evidence of the crosstalk between steroid hormone receptors and signal transduction pathways and in breast cancer, interaction between the ER and the EGFR has been well-documented (reviewed by Lichtner 2003, Johnston et al. 2003). Indeed, given that enhanced EGFR signalling may be a key adaptive change in the acquisition of endocrine-insensitive breast cancer, a combination of endocrine and anti-EGFR agents may prove to be more effective than either therapy alone and may delay the emergence of the endocrine-insensitive phenotype (Johnston et al. 2003, Lichtner 2003). In fact, we have shown that combination of tamoxifen and gefitinib in MCF-7 hormone-sensitive breast cancer cells has enhanced anti-proliferative effects and can delay the development of anti-hormone resistance (Gee et al. 2003). Currently, several Phase II trials have recently commenced in breast cancer evaluating the role of gefitinib in combination with anti-hormonal agents, including anastrazole (ArimidexTM), fulvestrant and tamoxifen.
A very interesting combination strategy of gefitinib with the anti-EGFR monoclonal antibody cetuximab has recently been evaluated. Although superficially both of these different classes of agents exert their effects by blocking ligand-induced EGFR phosphorylation and subsequent signal transduction, differences exist in some key mechanistic aspects of action. Monoclonal antibodies are associated with receptor internalization and degradation (Fan et al. 1994) and also inducing antibody-dependent cellular toxicity (Naramura et al. 1993), which further promotes antitumour action. Conversely, small-molecule TKIs can induce the formation of inactive EGFR homodimers and EGFR/HER-2 heterodimers thus additionally preventing HER-2 signalling (Moasser et al. 2001). The recent studies have shown that the inhibition of growth and the phosphorylation of EGFR and its downstream effector molecules Akt and MAPK were augmented in a panel of various cancer cell lines, when gefitinib and cetuximab were used in combination compared with either agent alone (Huang et al. 2004, Matar et al. 2004).
Conclusions
The complicated crosstalk between cellular components in cancer cells suggests that single blockade of EGFR by anti-EGFR agents or, indeed, prevention of any growth factor receptor signalling by its appropriate signal transduction inhibitor by monotherapy is unlikely to be sufficient for maximal antitumour activity as acquired or de novo resistance to signal transduction inhibitors is a common phenomenon. The identification of molecular markers including, as we believe, the IGF-IR, to aid the prediction of likely sensitivity to EGFR blockade and their additional blockade in combinatorial strategies, is essential to maximize patient benefit to these exciting new classes of anticancer agents.
Acknowledgements
Thanks to the Tenovus Tissue Culture Unit, Immunocytochemistry Unit, Sarah Razzaq and Sara Pumford for technical assistance, Lynne Farrow for statistical analysis and the Tenovus Cancer Charity for additional support. Funding was provided by Astra- Zeneca. H W was supported by a Wellcome Trust Vacation Scholarship. The authors declare the following potential conflicts of interest regarding this research: HE Jones, JMW Gee and RI Nicholson are in receipt of funding from AstraZeneca and RI Nicholson is also a member of an advisory board for AstraZeneca.
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2 Dip. Medicina Sperimentale e Diagnostica, Universita di Ferrara, Ferrara, Italy
This paper was presented at the 1st Tenovus/AstraZeneca Workshop, Cardiff (2005). AstraZeneca has supported the publication of these proceedings.
Abstract
Aberrant signalling through the epidermal growth factor receptor (EGFR) is associated with increased cancer cell proliferation, reduced apoptosis, invasion and angiogenesis. Over-expression of the EGFR is seen in a variety of tumours and is a rational target for antitumour strategies. Among the classes of agent targeting the EGFR are small-molecule inhibitors, which include gefitinib (IRESSATM), which acts by preventing EGFR phosphorylation and downstream signal transduction. De novo and acquired resistance, however, have been reported to gefitinib and here we describe evidence which indicates that the type II receptor tyrosine kinases (RTKs) insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (InsR) play important roles in the mediation of responses to gefitinib in the de novo- and acquired-resistance phenotypes in several cancer types. Moreover, combination strategies that additionally target the IGF-IR/InsR can enhance the antitumour effects of gefitinib.
Introduction
Advances in the understanding of the aberrant signalling pathways involved in the development and progression of cancer have resulted in the development of therapeutic agents or so-called smart drugs that target specific components of these pathways, the aim being that such precise targeting would hopefully produce greater activity and specificity against the tumour cell than is currently observed with traditional chemotherapeutic drugs. Based on these criteria, among the first and extremely successful targeted therapies in oncology could considered to be the development of anti-hormone therapies for breast and prostate cancer which blocked the action of the oestrogen receptor (ER) and androgen receptor respectively, with the majority of patients with hormone-responsive tumours showing good responses to this form of treatment (Griffiths et al. 1997, Gee et al. 2002). Over the last 20 years, advances in molecular diagnostics, complemented by the advent of high-throughput screening of potential anti-cancer compounds, has resulted in the development of novel drugs that can focus on targets with a specificity hitherto unavailable. This has been particularly evident in the emergence of inhibitors of specific signal transduction kinases, enzymes with critical roles in the pathways involved in the transfer of external stimuli such as hormones and growth factors to the nucleus. Thus, from this new genre of targeted anticancer therapies, notable successes include the humanized monoclonal antibody trastuzumab (HerceptinTM) for HER-2/neu-over-expressing metastatic breast cancer; the treatment of bcr/abl-translocation-positive chronic myelogenous leukaemia with the tyrosine kinase inhibitor (TKI) imatinib (GleevecTM); the use of cetuximab (ErbituxTM) in colorectal cancer; a chimeric monoclonal antibody that binds to the epidermal growth factor (EGF) receptor (EGFR); bevacizumab (AvastinTM), a humanized murine monoclonal antibody targeting the vascular endothelial growth factor and, finally, in the treatment of non-small cell lung carcinoma (NSCLC) the utilization of gefitinib and erlotinib (TarcevaTM), which are small-molecule EGFR TKIs (reviewed by Ross et al. 2004).
It is apparent, however, that despite the valuable role that these novel drugs may have in the treatment of malignancies, providing a much-needed therapeutic option for patients with advanced disease and often extremely poor prognosis, the clinical results overall have not equated with the expectations of such exciting specific therapies. Consequently, this article will focus on the smart strategies used to target a key player implicated in cancer development and disease progression, namely the EGFR, and discuss not only the successes but also the failures of these therapies, with particular focus on gefitinib in breast cancer. Furthermore, utilizing the current literature and work undertaken in our laboratory, we will describe mechanisms that may relate to a lack of response to gefitinib and postulate ways in which the efficacy and duration of response may be improved.
EGFR signalling
The EGFR belongs to the erbB receptor family which consist of four members: erbB1/HER-1/EGFR, erbB2/HER-2/neu, erbB3 and erbB4. The EGFR is a membrane glycoprotein that consists of a ligand-binding region, a transmembrane segment and an intracellular portion, the latter containing a tyrosine kinase domain upstream of additional autophosphorylation tyrosine sites within the C-terminus of the receptor (Gullick 2001). Activation of the EGFR occurs when ligands such as EGF, transforming growth factor- (TGF-) or amphiregulin bind to the EGFR causing receptor dimerization, either with another EGFR monomer or with another member of the erbB family. Dimerization subsequently activates the tyrosine kinase domains of each receptor resulting in the trans-autophosphorylation of the tyrosine sites on the other receptor molecule. These phosphorylated residues ultimately act as binding sites for adaptor proteins which recruit and activate a variety of signalling transduction cascades involved in cell proliferation and survival (Gullick 2001). Important pathways in erbB receptor signalling are the Ras/Raf/mitogen-activated protein kinase (MAPK) cascade, which is usually associated with cell proliferation, and the phosphoinositide 3-kinase (PI 3-kinase)/Akt route, which mediates numerous biological processes such as cell survival, gene expression and cell-cycle progression.
EGFR in cancer and EGFR-targeted strategies
The EGFR is expressed in a wide range of solid tumours including breast and prostate and has been implicated as playing a major role in tumour growth and progression (Salomon et al. 1995, Nicholson et al. 2001a). Moreover, many solid tumours also demonstrate elevated expression of the cognate ligands for EGFR, namely EGF and TGF- (Salomon et al. 1995). It has also been shown in breast cancer that lack of response to endocrine therapy, together with poor survival and increased metastasis, is associated with over-expression of EGFR (Nicholson et al. 1994, 2001b). Additionally, in vitro studies have demonstrated that transfection of EGFR into hormone-dependent breast tumour cells can mediate acquired resistance to the anti-oestrogen therapy tamoxifen (van Agthoven et al. 1992), such resistance being a major problem in the treatment of clinical breast cancer (Gee et al. 2002). Furthermore, we have demonstrated in our laboratory that acquired resistance to the pure anti-oestrogen fulvestrant (FaslodexTM) and tamoxifen (NolvadexTM) can be facilitated by EGFR-mediated growth pathways. These resistant cell lines show increased EGFR expression/signalling and are highly sensitive to the EGFR TKI gefitinib (McClelland et al. 2001, Knowlden et al. 2003). Similarly to breast cancer, aberrant EGFR signalling has been linked to the progression of hormone-responsive prostate cancer to the hormonerefractory state (Djakiew 2000).
Clearly, the EGFR represents an important target in cancer therapy and this has prompted the development of a variety of different agents to prevent EGFR signal transduction. These agents have been reviewed elsewhere (Ciardiello & Tortora 2001) but briefly, the most advanced in clinical development include small-molecule TKIs such as gefitinib and erlotinib, both of which are quinazolines and act by competitively inhibiting ATP binding to the tyrosine kinase domain of the EGFR, thus preventing EGFR autophosphorylation and subsequent downstream signal transduction, and the monoclonal antibodies cetuximab and ABX-EGF, which bind to the external domain of the EGFR. Indeed, both gefitinib and erlotinib have now been approved for the treatment of advanced NSCLC and cetuximab has been approved for advanced colorectal cancer.
Gefitinib: preclinical and clinical evaluation
Preclinical studies have indicated that gefitinib is growth-inhibitory to a range of human tumour xenografts and, importantly, potentiates the activity of various cytotoxic agents when used in combination studies (Ciardiello et al. 2000, Wakeling et al. 2002). Evidence shows that gefitinib was growth-inhibitory in xenografts of both ER-positive and ER-negative ductal carcinoma in situ tissue (Chan et al. 2002) and, furthermore, the inhibitor could restore tamoxifen sensitivity in de novo resistant HER-2-overexpressing MCF-7 human breast cancer xenografts (Massarweh et al. 2002). Gefitinib was extremely effective at preventing EGFR autophosphorylation and the subsequent downstream activation of extracellular- signal-regulated kinase (ERK) 1/2 MAPK and Akt in our breast cancer models of tamoxifen or fulvestrant resistance, under both basal and EGF-ligand- primed conditions (McClelland et al. 2001, Nicholson et al. 2001b, 2004, Knowlden et al. 2003). In addition, cell growth of each model was inhibited dramatically (90%), which was in marked contrast to the minimal effect (<10%) that the inhibitor displayed on the growth of the parental MCF-7 cells (McClelland et al. 2001, Nicholson et al. 2001b, 2004, Knowlden et al. 2003). Overall, such data indicate that gefitinib may be valuable in the treatment of EGFR expressing ER-negative and ER-positive breast tumours.
The clear rationale for the use of gefitinib in breast cancer has given rise to numerous clinical trials with the inhibitor in this cancer type. Indeed, gefitinib is currently undergoing evaluation in breast cancer in a range of Phase II studies. Excitingly, we have shown, in a Phase II study, that gefitinib produced worthwhile clinical benefit in ER-positive patients with acquired tamoxifen resistance, with 11 out of 13 patients demonstrating partial responses and disease stabilization, as well as an extended median time to progression (Gee et al. 2004, Gutteridge et al. 2004). Additionally, another Phase II trial assessing the efficacy of gefitinib monotherapy in patients with advanced breast cancer showed that 10 out of 33 patients demonstrated stable disease (Baselga et al. 2003).
EGFR status and response to gefitinib
It has become apparent however that although clinical benefit has been observed with gefitinib in numerous solid tumour types reported to over-express EGFR including breast, the clinical data have also disappointingly revealed the existence of de novo and acquired resistance to gefitinib therapy with some patients deriving no clinical benefit or displaying varying durations of response prior to disease relapse (Baselga et al. 2002, Ranson 2002, Ranson et al. 2002, Kelly & Averbuch 2004). Critically, it has now been established that although there is a strong correlation between EGFR levels and aggressive disease with reduced survival (Nicholson et al. 2001b), a clear association between EGFR expression/phosphorylation and predicted response to various EGFR-targeted agents including gefitinib does not exist (reviewed by Arteaga 2002). Moreover, in our clinical study with gefitinib, in advanced breast cancer patients who were either ER-positive with acquired tamoxifen resistance or who had de novo hormone-resistant ER-negative disease, the gefitinib-responsive patients were in fact the former group who expressed only modest EGFR prior to and after 8 weeks gefitinib treatment. Surprisingly, 85% of the ER-negative/over-expressing EGFR patients had de novo resistance to gefitinib (Gee et al. 2004). Interestingly, this equates with our in vitro model system data for ER-positive acquired tamoxifen resistance, where we have shown conclusively that modestly increased EGFR signalling is growth-contributory (Knowlden et al. 2003). A proportion of these responding patients (five out of nine) also showed an obvious fall in phosphorylated EGFR and activated MAPK (Gee et al. 2004); however, decreases in EGFR phosphorylation were not observed in all patients showing clinical benefit (Gee et al. 2004), suggesting that a non-classical gefitinib-response mechanism may operate in some patients.
Such observations have been noted in other cancer types. For example, EGFR is over-expressed in up to 70% of NSCLC (Rusch et al. 1997, Fontanini et al. 1998) yet only modest objective responses of between 12 and 18% were observed with gefitinib in Phase II trials in this cancer type (Fukuoka et al. 2003, Kris et al. 2003). Moreover, from these trials, it has been determined that EGFR membrane staining predicts neither tumour response nor symptom improvement in NSCLC (Bailey et al. 2003). Interestingly however, recent research has identified specific somatic mutations in the EGFR tyrosine kinase domain in a subset of NSCLC patients who showed a dramatic improvement from gefitinib (Lynch et al. 2004, Paez et al. 2004), which may aid patient selection. It has also been observed that responses to another anti- EGFR agent, namely erlotinib, in patients with NSCLC did not show any correlation to EGFR staining (Perez-Soler et al. 2004). It is therefore abundantly clear that considerable research is required to not only define the mechanisms which promote both acquired and de novo resistance to EGFR inhibitors with the aim of improving efficacy and duration of response in a greater number of patients but additionally, delineate other molecular markers which may predict sensitivity to EGFR inhibitors such as gefitinib.
Characteristics of acquired resistance to gefitinib
Up-regulation of the type II receptor tyrosine kinase (RTK) insulin-like growth factor-I receptor (IGF-IR) signalling
The varying durations of response (3–12 months) displayed by responders to gefitinib in various different cancer types (Barton et al. 2001, Baselga et al. 2002, Ranson 2002, Ranson et al. 2002, Douglass 2003, Schiller 2003, Kelly & Averbuch 2004) indicated the fairly rapid acquisition of resistance to the TKI. In our laboratory, we have generated several model systems in breast, prostate and lung cancer to study this phenomenon in vitro. For example, utilizing our in vitro model system for acquired tamoxifen resistance, which consists of EGFR-positive, MCF-7- derived, tamoxifen-resistant breast cancer cells (Knowlden et al. 2003), we continuously exposed this cell line, designated TAM-R, to 1 μM gefitinib, a concentration shown to be growth-inhibitory to the TAM-R cells (Knowlden et al. 2003). A sustained growth inhibition (90%) was observed for 4 months before the surviving cells resumed proliferation and a stable gefitinib-resistant subline (TAM/TKI-R) was established after a further 2 months, which showed no detectable basal phosphorylated EGFR activity and minimal MAPK activity (Jones et al. 2004a). Compared with the parental TAM-R cells, however, the TAM/TKI-R cells demonstrated elevated levels of phosphorylated IGF-IR and, furthermore, an increased sensitivity to growth inhibition by the IGF-IR TKI, AG1024 (Calbiochem, Merck Biosciences, Nottingham, UK; Jones et al. 2004a).
Over-expression and activation of the IGF-IR, a transmembrane tyrosine kinase receptor, and its downstream signalling molecules have been linked to disease progression in breast cancer and other cancer types (Yu & Rohan 2000). In addition, the TAM/TKI-R cell line also showed elevated levels of activated Akt and protein kinase C (PKC) , which could be modulated by treatment with AG1024, suggesting that these components were downstream targets for IGF-IR signalling in these cells (Jones et al. 2004a). Moreover, preliminary data from another of our MCF-7-derived breast cancer cell lines dually resistant to the pure-anti-oestrogen faslodex and gefitinib (FAS/TKI-R) indicates that compared with their faslodex-resistant parents (FASR) (McClelland et al. 2001), the FAS/TKI-R cells also demonstrate elevated expression and activation of IGF-IR and PKC (HE Jones, JMW Gee, D Barrow, M Rubin & RI Nicholson; unpublished observation), the significance of which is currently under investigation.
Overall, such findings indicate that IGF-IR is an important therapeutic target in acquired gefitinib resistance in breast cancer and strategies which target this receptor may increase the efficacy and duration of response to gefitinib. A crucial question is, however, whether IGF-IR signalling is important in mediating gefitinib responses in other cancer types. To this end, we have shown additionally that androgen-independent prostate cancer cells with acquired gefitinib resistance also show a dramatic marked increase in the expression and activation of membrane-associated IGF-IR, together with considerable increased levels of the ligand IGF-II and, furthermore, a dependence on IGF-IR for growth (Jones et al. 2004a), thus supporting the importance of IGF-IR signalling in the acquired-gefitinib-resistance phenotype. Indeed, evidence is accumulating which indicates that an association exists between elevated IGF-IR expression/signalling and its downstream components such as Akt and resistance to drugs which inhibit erb-family signal transduction in various cancers. For example, IGF-IR via PI 3-kinase/Akt activation has been shown to mediate resistance to the anti-EGFR monoclonal antibody 225 in glioblastoma cells (Chakravati et al. 2002) and also the selective EGFR TKI AG1478 (Sigma Chemical Co., St Louis, MO, USA) in the DiFi human colorectal cancer cell line (Liu et al. 2001).
Elevated HER-2 (erbB-2, neu) signalling
It is established that the erbB receptor HER-2 is amplified and over-expressed in approximately 30% of breast cancers and that, furthermore, such overexpression is associated with aggressive metastatic disease with reduced time to relapse (Slamon et al. 1987). A ligand for HER-2 has not been clearly defined; however, through heterodimerization HER-2 can mediate the signal transduction of all other erbB family members when they bind their cognate ligands (Gullick 2001). Interestingly, we have shown that in our tamoxifen-resistant breast cancer cells the expression of both EGFR and HER-2 is increased but, moreover, HER-2 preferentially dimerizes with EGFR in these cells which subsequently show good responses to gefitinib (Knowlden et al. 2003) and the HER-2 inhibitor trastuzumab (Fig. 1A). Once these cells acquire resistance to gefitinib, however, levels of HER- 2 expression and activity are further increased (Fig. 1B) but, paradoxically, sensitivity to trastuzumab is lost (Fig. 1A). Thus, gefitinib resistance has generated cross-resistance to another agent, namely trastuzumab. Interestingly, IGF-IR signalling has also been implicated in modulating the responses to trastuzumab (Lu et al. 2001). In addition, it has also been observed that IGF-IR can unidirectionally activate HER-2, which involves a physical association of the two receptors (Balana et al. 2001). Our gefitinib-resistant breast cancer cells also show evidence of the existence of a physical interaction between the IGF-IR and HER-2, which furthermore can co-localize in areas at the tumour cell membranes (Fig. 2A and B). Additionally, preliminary work excitingly shows that concentrations of the IGF-IR TKI AG1024 that block IGF-IR phosphorylation also inhibited HER-2 activity (Fig. 2C). Interestingly, other workers have shown that the IGF-IR-mediated attenuation of trastuzumab growth inhibition is dependent upon signalling via PI 3-kinase/Akt and not MAPK in HER-2 over-expressing breast cells (Lu et al. 2004). Similarly, we have also shown that in our gefitinib-resistant breast cancer cells, which have elevated HER-2 and IGF-IR activity, Akt phosphorylation is increased and that IGF-IR signalling appears to be dissociated from MAPK (Jones et al. 2004a). Such data adds further to the observation that the IGF-IR is increasingly becoming a key therapeutic target to potentially improve the efficacy of erbB inhibitors.
Maintenance of ER phosphorylation in acquired gefitinib-resistant breast cancer
In our TAM/TKI-R acquired-resistance gefitinib model, ER expression is retained at levels comparable to the parental TAM-R and MCF-7 cells and, furthermore, this ER appears functional, since the anti-oestrogen fulvestrant decreases the growth of the TAM/TK1-R cells and these cells express increased oestrogen-regulated gene levels (HE Jones, M Giles, JMW Gee, D Barrow, AE Wakeling & RI Nicholson; unpublished observation). Interestingly, the ER appears to be highly phosphorylated at serine-118 in such cells, its activity even exceeding the high level observed in their TAM-R parents (Britton et al. 2002). Moreover, this occurs despite lower levels of activated MAPK, the kinase implicated in phosphorylation of this site in the parental TAM-R cells (Britton et al. 2003). Intriguingly, we have shown using immunopreciptation and immunofluorescence that activated PKC, significantly elevated in TAM/TKI-R and a downstream target for IGF-IR (Jones et al. 2004a), can physically interact with and co-localize with activated nuclear ER. Moreover, the PKC inhibitor bisindoylmaleimide IX (Ro-31-8220; Calbiochem) blocks serine-118 ER phosphorylation and reduces expression of ER-regulated genes, suggesting productive crosstalk between ER and PKC in these gefitinib resistant cells (HE Jones, M Giles, JMW Gee, D Barrow, AE Wakeling & RI Nicholson; unpublished observation). In summary, these data suggest that increased IGF-IR-driven, nuclear-activated PKC may contribute to growth of acquired gefitinib-resistant breast cancer cells via its recruitment to and phosphorylation of ER to promote cell growth. Interestingly, our additional acquired gefitinib-resistant breast cancer model, derived from FASR breast cancer cells, shows very low levels of ER but is still phosphorylated and is coupled with increased expression of oestrogen-regulated genes compared with the parental cells. Since these cells also show increased IGF-IR/nuclear PKC signalling versus their parental cells, it is possible that the PKC/ER crosstalk mechanism may again be contributory.
Importance of type II RTKs in partial or de novo gefitinib resistance
It is known that EGFR signalling can be modulated by several mechanisms which include heterodimerization with other members of the EGFR family, such as HER-2 (erbB-2; Gullick 2001), and transactivation by, or crosstalk with, other growth factor receptors such as IGF-IR (Roudabush et al. 2000, Wang et al. 2002) or steroid hormone receptors (Lichtner 2003). Indeed, we have shown that in our tamoxifen-resistant breast cancer model the IGF-IR appears to be permissive for EGFR signalling (Nicholson et al. 2004). Importantly, we have demonstrated that similarly to acquired gefitinib resistance in breast and prostate cancer, the efficacy of gefitinib may be compromised in de novo resistance in colorectal cancer cells by the involvement of the type II RTK insulin receptor (InsR) isoform-A (InsR-A; Jones et al. 2004b), a closely related family member to the IGF-IR and which has been linked to cancer development and disease progression (reviewed by Denley et al. 2003). Indeed, data indicated that, via EGFR blockade, gefitinib can facilitate the activity of the InsR which in turn can modulate EGFR phosphorylation (Jones et al. 2004b). Excitingly, preliminary work in our laboratory also suggests a major role for the involvement of the IGF-IR in the partial resistance to gefitinib displayed by NSCLC cells. It is also feasible that in tumour cells that, are insensitive or only partially responsive to gefitinib, existing high IGF-IR/InsR activity may also play an important role in the maintenance of cell proliferation independently of the EGFR.
Strategies to improve gefitinib response with other signal transduction inhibitors
Targeting the IGF-IR
Following the elucidation of the mechanisms underlying the acquired and de novo resistance to gefitinib, combination strategies involving other signal transduction inhibitors which inhibit these resistance mechanisms would hopefully extend durations of response and improve the efficacy of gefitinib. It is clear that the type II RTKs, in particular the IGF-IR, represent important targets in the search to improve gefitinib response not only in breast and prostate cancer but also in other cancer types. Indeed, numerous pharmaceutical programmes have been implemented to develop specific inhibitors of the IGF-IR but this is still in the early stages of discovery; however, potential anti-IGF-IR strategies have been reviewed by Surmacz (2003). We have already shown that the acquisition of gefitinib resistance in our tamoxifen-resistant breast cancer cells can be delayed or even prevented by the combination of gefitinib plus an IGF-IR inhibitor (Nicholson et al. 2004) and, excitingly, early studies indicate that in the presence of an IGF-IR inhibitor gefitinib effects can be observed on tumour cells that had previously demonstrated de novo resistance to this inhibitor.
Targeting other elements in combination with gefitinib
There is considerable evidence of the crosstalk between steroid hormone receptors and signal transduction pathways and in breast cancer, interaction between the ER and the EGFR has been well-documented (reviewed by Lichtner 2003, Johnston et al. 2003). Indeed, given that enhanced EGFR signalling may be a key adaptive change in the acquisition of endocrine-insensitive breast cancer, a combination of endocrine and anti-EGFR agents may prove to be more effective than either therapy alone and may delay the emergence of the endocrine-insensitive phenotype (Johnston et al. 2003, Lichtner 2003). In fact, we have shown that combination of tamoxifen and gefitinib in MCF-7 hormone-sensitive breast cancer cells has enhanced anti-proliferative effects and can delay the development of anti-hormone resistance (Gee et al. 2003). Currently, several Phase II trials have recently commenced in breast cancer evaluating the role of gefitinib in combination with anti-hormonal agents, including anastrazole (ArimidexTM), fulvestrant and tamoxifen.
A very interesting combination strategy of gefitinib with the anti-EGFR monoclonal antibody cetuximab has recently been evaluated. Although superficially both of these different classes of agents exert their effects by blocking ligand-induced EGFR phosphorylation and subsequent signal transduction, differences exist in some key mechanistic aspects of action. Monoclonal antibodies are associated with receptor internalization and degradation (Fan et al. 1994) and also inducing antibody-dependent cellular toxicity (Naramura et al. 1993), which further promotes antitumour action. Conversely, small-molecule TKIs can induce the formation of inactive EGFR homodimers and EGFR/HER-2 heterodimers thus additionally preventing HER-2 signalling (Moasser et al. 2001). The recent studies have shown that the inhibition of growth and the phosphorylation of EGFR and its downstream effector molecules Akt and MAPK were augmented in a panel of various cancer cell lines, when gefitinib and cetuximab were used in combination compared with either agent alone (Huang et al. 2004, Matar et al. 2004).
Conclusions
The complicated crosstalk between cellular components in cancer cells suggests that single blockade of EGFR by anti-EGFR agents or, indeed, prevention of any growth factor receptor signalling by its appropriate signal transduction inhibitor by monotherapy is unlikely to be sufficient for maximal antitumour activity as acquired or de novo resistance to signal transduction inhibitors is a common phenomenon. The identification of molecular markers including, as we believe, the IGF-IR, to aid the prediction of likely sensitivity to EGFR blockade and their additional blockade in combinatorial strategies, is essential to maximize patient benefit to these exciting new classes of anticancer agents.
Acknowledgements
Thanks to the Tenovus Tissue Culture Unit, Immunocytochemistry Unit, Sarah Razzaq and Sara Pumford for technical assistance, Lynne Farrow for statistical analysis and the Tenovus Cancer Charity for additional support. Funding was provided by Astra- Zeneca. H W was supported by a Wellcome Trust Vacation Scholarship. The authors declare the following potential conflicts of interest regarding this research: HE Jones, JMW Gee and RI Nicholson are in receipt of funding from AstraZeneca and RI Nicholson is also a member of an advisory board for AstraZeneca.
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