CONFERENCE PAPER
Endocrine-Related Cancer (2006) 13 S89–S97
Inductive mechanisms limiting response to anti-epidermal growth factor receptor therapy Iain R Hutcheson, Janice M Knowlden, Helen E Jones, Rajpal S Burmi, Richard A McClelland, Denise Barrow, Julia M W Gee and Robert I Nicholson Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK (Requests for offprints should be addressed to I R Hutcheson; Email:
[email protected])
Abstract Aberrant epidermal growth factor receptor (EGFR) signalling, a key feature of a variety of human malignancies, can drive a range of mechanisms underlying tumour growth and progression, including increased cell proliferation, angiogenesis, metastasis and decreased apoptosis. AntiEGFR therapies, as monotherapies and in combination with chemotherapy, have proved effective in inhibiting these processes both in the clinical and in the preclinical settings. However, only a small cohort of patients have derived significant benefit from this therapy, with both de novo and acquired resistance to these agents evident in a number of recent studies. If we are to improve the effectiveness of such targeted therapies, then there is an urgent need to understand the resistance mechanisms. Here, we describe both non-genomic and genomic mechanisms of resistance to the selective EGFR tyrosine kinase inhibitor gefitinib (IRESSA), which we have identified initially in an EGFR-positive tamoxifen-resistant MCF-7 breast cancer cell line, but more recently in other EGFR-positive cancer types. Importantly, we show that gefitinib, in common with anti-hormonal agents, is not a passive bystander in the cellular response to drug treatment, but plays an active role in promoting signalling pathways that serve to limit its anti-tumour activity and maintain the cellular cohort from which acquired resistance can ultimately evolve. These findings indicate that inductive signalling is an important determinant of response to EGFR-targeted therapies and deciphering such pathways may provide us with the opportunity to design more effective strategies to combat resistance mechanisms and improve response to initial therapy. Endocrine-Related Cancer (2006) 13 S89–S97
Introduction The epidermal growth factor receptor (EGFR), a member of the c-erbB receptor tyrosine kinase family, is a membrane glycoprotein composed of an extracellularbinding domain, a transmembrane domain containing a single hydrophobic anchor sequence and an intracellular domain containing tyrosine kinase activity (Olayioye et al. 2000, Schlessinger 2000). Activation of EGFR results from the binding of (EGF)-related growth factors, such as EGF, TGFa and amphiregulin, which induce receptor homo- and/or heterodimerization and This paper was presented at the 2nd Tenovus/AstraZeneca Workshop, Cardiff (2006). AstraZeneca supported the meeting and the Welsh School of Pharmacy, Cardiff University has supported the publication of these proceedings.
stimulation of the intrinsic receptor tyrosine kinase activity. This promotes autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptor, providing docking sites for a variety of adaptor proteins and enzymes involved in the recruitment and activation of downstream intracellular-signalling cascades, including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3K) pathways (Schlessinger 2000). These signalling cascades can promote proliferation, angiogenesis and invasion and inhibit apoptosis, key mechanisms underlying tumour growth and progression (Salomon et al. 1995). This oncogenic potential in conjunction with the aberrant expression and/or activation of EGFR, which has been reported in a wide range of human malignancies, including non-small cell lung carcinoma, breast, prostate,
Endocrine-Related Cancer (2006) 13 S89–S97 1351–0088/06/013–S89 q 2006 Society for Endocrinology Printed in Great Britain
DOI:10.1677/erc.1.01279 Online version via http://www.endocrinology-journals.org
I R Hutcheson et al.: Gefitinib-induced signalling mechanisms colorectal and head and neck cancers, provides a strong rationale for targeting this growth factor receptor (Nicholson et al. 2001, Baselga 2002).
Targeting the EGFR: preclinical and clinical data with gefitinib Two major classes of agent have been developed to inhibit EGFR activity, monoclonal antibodies, such as cetuximab (Erbitux, C225), which bind the extracellular ligand-binding domain of the receptor and small molecule tyrosine kinase inhibitors, such as gefitinib (Iressa, ZD1839) and erlotinib (Tarceva, OSI-774; Baselga & Arteaga 2005). Gefitinib [4-(3-chloro-4fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy) quinazoline] is a synthetic quinazolone derivative that selectively inhibits the EGFR tyrosine kinase in a reversible manner (Wakeling et al. 2002). It has been clearly established in the preclinical studies that gefitinib potently inhibits growth in a range of human cancer cell lines that express a functional EGFR, including breast, prostate, ovarian, colon and nonsmall cell lung (Ciardiello & Tortora 2001). Furthermore, similar growth-inhibitory effects of gefitinib have been reported in a variety of human xenograft models (Wakeling et al. 2002). Other in vitro models have also demonstrated pro-apoptotic, anti-angiogenic and anti-metastatic activity with this EGFR-targeted agent (Ciardiello et al. 2001, Ciardiello & Tortora 2001, Hiscox et al. 2004). Gefitinib has also been shown to have potential use in combination with conventional chemotherapy and radiotherapy, where it has been shown to enhance the effects of both cytotoxic agents and ionizing radiation (Ciardiello et al. 2000, Raben et al. 2002). This clear therapeutic promise in the preclinical setting has resulted in gefitinib undergoing extensive clinical trials in a variety of EGFRpositive solid tumour types, principally non-small cell lung carcinoma and also prostate, breast, ovarian, head and neck and colorectal cancers (Cappuzzo et al. 2006). These trials have demonstrated that gefitinib is well tolerated and can provide significant clinical benefit in a small number of patients. However, overall the data have proved quite disappointing with a large number of patients either deriving no clinical benefit or suffering disease relapse following an initial, quite variable, responsive phase (Kelly & Averbuch 2004, Jones et al. 2005, Ramsay Camp et al. 2005, Cappuzzo et al. 2006). These findings clearly indicate an urgent need to understand the mechanisms responsible for driving both de novo and acquired gefitinib resistance, if we are to improve the efficacy and duration of response to this and other EGFR-targeted agents. S90
Elevated PI-3K/AKT signalling is a key feature of gefitinib resistance A range of possible resistance mechanisms to antiEGFR agents have been identified both in the preclinical and in the clinical studies and include receptor mutation (Riedel & Febbo 2005) and activation of alternative oncogenic-signalling pathways (Camp et al. 2005). Recent evidence has identified the PI-3K/AKT-signalling pathway as a potential mediator of resistance to anti-growth factor therapies, including those targeting EGFR (Chakravarti et al. 2002, Bianco et al. 2003, She et al. 2003, Jones et al. 2004, Camirand et al. 2005). Elevated levels of AKT have been shown to be a feature of de novo and acquired gefitinib resistance in a range of human cancer cell lines, including glioblastoma, breast, non-small cell lung and prostate (Chakravarti et al. 2002, Bianco et al. 2003, Li et al. 2003, She et al. 2003, Jones et al. 2004, Camirand et al. 2005, Festuccia et al. 2005, Ihle et al. 2005). Two mechanisms have been implicated in mediating elevated AKT activity in gefitinib resistance, constitutive activation of the pathway due to loss of phosphatase and tensin homologue (PTEN) activity and activation by an alternative growth factor receptor tyrosine kinase, the insulin-like growth factor type I receptor (IGF-IR). The main function of PTEN is to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate, the lipid second messenger that binds and activates AKT, reducing its levels within the cell (Cantley & Neel 1999, Simpson & Parsons 2001). Consequently, PTEN acts in opposition to PI-3K which drives production of this lipid messenger. Therefore, loss of PTEN function leads to accumulation of phosphatidylinositol 3,4,5-trisphosphate in the cell membrane and constitutive activation of AKT (Cantley & Neel 1999, Simpson & Parsons 2001). Elevated expression of phosphorylated AKT due to functional loss of PTEN has been identified as the central mechanism mediating resistance to the anti-proliferative and pro-apoptotic actions of gefitinib in MDA-468 breast cancer cells (Bianco et al. 2003, She et al. 2003). Furthermore, re-introduction of wild-type PTEN into these cells reduces AKT activity and restores sensitivity to gefitinib (Bianco et al. 2003, She et al. 2003). The IGF-IR is a member of the type II receptor tyrosine kinase family, which also includes the insulin receptor (Ullrich et al. 1986) and has been linked to disease progression and recurrence in clinical breast cancer (Rocha et al. 1997, Turner et al. 1997). Ligand binding of insulin, insulin-like growth factor I or II (IGF-I or IGF-II) leads to receptor autophosphorylation www.endocrinology-journals.org
Endocrine-Related Cancer (2006) 13 S89–S97 and subsequent phosphorylation of substrate proteins, primarily the insulin receptor substrate-1 (IRS-1; White 1997). IGF-IR signalling via the PI-3K/AKT pathway has been shown to mediate resistance to the anti-c-erbB2 monoclonal antibody trastuzumab in SKBR3 and c-erbB2-transfected MCF-7 breast cancer cell lines (Lu et al. 2001, 2004). More recently, increased IGF-IR-mediated AKT-signalling activity has also been implicated in the development of resistance to the selective EGFR tyrosine kinase inhibitor, AG1478, in glioblastoma cells (Chakravarti et al. 2002) and also to the anti-EGFR monoclonal antibody 225 in the DiFi human colorectal cancer cell line (Liu et al. 2001). A role for IGF-IR has also been implicated in gefitinib resistance with overexpression of IGF-IR in SKBR3 breast cancer cells significantly reducing sensitivity of these cells to the growthinhibitory actions of this anti-EGFR therapy (Camirand et al. 2005). Furthermore, in our own laboratory, we have generated an acquired gefitinibresistant MCF-7 breast cancer cell line through continuous exposure of EGFR-positive tamoxifenresistant MCF-7 cells to gefitinib, at a concentration previously shown to be growth inhibitory to these cells (Knowlden et al. 2003). Following a potent and sustained growth inhibition, lasting approximately 4 months, regrowth of surviving cells is observed and a stable gefitinib-resistant subline (TAM/TKI-R) established after a further 2 months (Jones et al. 2004). This gefitinib-resistant cell line shows no detectable basal phosphorylated EGFR activity and minimal MAPK activity, but significantly elevated levels of phosphorylated IGF-IR and AKT compared with the parental Tam-R cells (Jones et al. 2004). Targeting the IGF-IR with the selective tyrosine kinase inhibitor, AG1024, reduces phosphorylated AKT levels and potently inhibits growth of the TAM/TKI-R cells indicative of a central role for IGF-IR in mediating the acquisition of resistance to gefitinib in this cell line (Jones et al. 2004).
Residual PI-3K/AKT signalling is a feature of gefitinib response An essential event for resistant growth is that a cohort of tumour cells initially evades inhibition during the drug-responsive phase. Deciphering the mechanisms underlying how tumour cells are apparently protected from maximal inhibition during early treatment is required if we are to design intelligent therapies to further improve initial response and thwart development of resistance. We have recently shown that antihormones, such as tamoxifen and faslodex, are potent www.endocrinology-journals.org
anti-proliferative agents, but elicit only a partial proapoptotic response in ER-positive MCF-7 breast cancer cells see in this issue, Gee et al. (2003). This incomplete response to anti-hormonal therapy is associated with residual, downstream activity of the proliferation and cell survival kinases, MAPK and AKT, allowing a cohort of cells to evade inhibition during the initial drug-responsive phase (see in this issue, Gee et al. (2006)). We have now established that residual downstream kinase activity, mediating early cell survival and resistance, applies equally to antigrowth factor therapies, such as gefitinib. We have found that in tamoxifen-resistant MCF-7 breast cancer cells (Tam-R), which are highly dependent on EGFR signalling for growth (Knowlden et al. 2003), gefitinib treatment results in considerable anti-proliferative activity but incomplete pro-apoptotic effects in responsive cells. This is reflected by the effects of gefitinib on downstream-signalling pathway activity, with a potent reduction of phosphorylated MAPK levels, but an incomplete blockade of AKT-signalling activity, being observed following acute treatment of Tam-R cells with this agent (Knowlden et al. 2003, Jordan et al. 2004). Similar residual AKT activity has been reported in a variety of breast and non-small cell lung cancer cell lines following acute gefitinib treatment (Janmaat et al. 2003, Ono et al. 2004, Camirand et al. 2005). Surprisingly, we have recently found that when gefitinib treatment of Tam-R cells is further extended to 7 days, although EGFR and MAPK activity and Tam-R growth continue to be potently inhibited, levels of phosphorylated AKT are not only recovered but also significantly enhanced compared with untreated control cells (Knowlden et al. 2006). Furthermore, this more prolonged exposure to gefitinib also potentiates IGF-II-induced activation of AKT, suggesting that this residual signalling is a consequence of IGF-IR-signalling activity (Knowlden et al. 2006).
EGFR recruits IRS-1 in EGFR-positive cancer cell lines The ability of gefitinib to promote IGF-IR signalling in our Tam-R cell line would suggest the existence of a novel cross-talk mechanism whereby EGFR is capable of regulating IGF-IR pathway activity. Further evaluation of this potential cross-talk mechanism has revealed that the EGFR has no direct effect on IGFIR expression or phosphorylation levels in Tam-R cells; however, EGFR is capable of cross-talking with another component of the IGF-IR-signalling cascade, the adaptor protein IRS-1. Classically, IRS-1 has been S91
I R Hutcheson et al.: Gefitinib-induced signalling mechanisms identified as an adaptor protein for the type II receptor tyrosine kinase family, however, it has also been shown to interact with other key proteins, such as integrins (Vuori & Ruoslahti 1994), cadherins (Hellawell et al. 2002) and steroid hormone receptors (Mauro et al. 2003). Following its recruitment by IGF-IR, IRS-1 can be phosphorylated at a number of tyrosine residues providing docking sites for SH2-containing proteins (White 1997). Two key residues which when phosphorylated play a central role in recruitment of important downstream signal transduction cascades are tyrosine 612 (Y612) and tyrosine 896 (Y896; White 1997, Esposito et al. 2001, Hers et al. 2002). Phosphorylation of IRS-1 at Y612 has been shown to act as a docking site for the p85-regulatory subunit of PI-3K which when activated serves to drive AKT activity (Esposito et al. 2001). This is the principal pathway recruited by phosphorylated IRS-1 in a number of cell systems. Phosphorylation of IRS-1 at Y896 acts as a recruitment site for the adaptor protein Grb2 that is involved in triggering the MAPK-signalling pathway (White 1997, Hers et al. 2002). Identification of EGFR as being a novel IRS-1interacting protein in Tam-R MCF-7 breast cancer cells arises from the finding that, under basal growth conditions (e.g. in the absence of exogenous growth factors), IRS-1 is highly phosphorylated on residue Y896 and this activity can be further promoted by EGF treatment in these cells. In contrast, treatment of Tam-R cells with IGF-II promotes Y612, but not Y896, phosphorylation of IRS-1. Therefore, EGFR appears to be capable of recruiting IRS-1 as part of its mechanism to engage the MAPK-signalling cascade in this cell line. Indeed, we were able to confirm that EGF-induced phosphorylation of IRS-1 at Y896 results from a direct association of EGFR with this adaptor protein using immunoprecipitation/western-blotting techniques (Knowlden et al. 2006). The ability of EGFR to recruit IRS-1 is a novel signalling phenomenon that has not previously been described to date in breast cancer cells. Interestingly, we found that such a phenomenon is not unique to tamoxifen-resistant MCF-7 breast cancer cells as we have gone on to demonstrate that EGF also promotes phosphorylation of IRS-1 at Y896 in a range of EGFR-positive cancer cell lines, namely T47D breast cancer cells, DU145 and LNCaP prostate cancer cells and A549 non-small cell lung carcinoma cells (Knowlden et al. 2006). Furthermore in LNCaP cells, this effect of EGF is again a result of a direct association of EGFR with IRS-1. In support of these findings, EGF-dependent IRS-1 phosphorylation has also been reported in human epidermoid carcinoma S92
A431 cells and in primary cultures of rat hepatocytes (Fujioka et al. 2001, Fujioka & Ui 2001). It is not entirely surprising that EGFR can bind IRS-1. A potential interaction between EGFR and IRS-1 has been predicted from the binding of peptides, representing the physical sites of EGFR tyrosine phosphorylation, to protein microarrays comprising all Src homology 2 and phosphotyrosine-binding domains encoded in the human genome (Jones et al. 2006). Furthermore, the phosphorylated NPXY motifs in activated insulin and IGF-IR receptors to which the phosphotyrosine-binding domains of IRS molecules bind are also present in the C-terminus region of EGFR (Songyang et al. 1995). Indeed, the presence of all three of these NPXY present in EGFR were found to be indispensable for IRS-1 to be tyrosine phosphorylated in response to EGF in EGFR-transfected Chinese hamster ovary cells (Fujioka et al. 2001). As previously mentioned, the principal phosphorylated form of IRS-1 in Tam-R cells is Y896. Since EGFR is the prime mediator of IRS-1 Y896 phosphorylation in Tam-R cells, this observation would suggest that EGFR is the dominant recruiter of IRS-1 in this cell line. In support of this concept, we have gone on to demonstrate when Tam-R cells are treated with EGF and IGF-II in combination, EGFR/IRS-1 association and IRS-1 phosphorylation at Y896 is maintained, while IGF-IR/IRS-1 association and Y612 phosphorylation of IRS-1 are considerably reduced (Knowlden et al. 2006). Therefore, the association of IRS-1 with EGFR prevents recruitment of IRS-1 by IGF-IR which serves to actively limit signalling via this receptor while further promoting the EGFR/MAPK pathway that is central to Tam-R cell growth. Interestingly, the suppression of IGF-IR signalling by EGFR has also been reported in a prostate epithelial cell line, CPTX 1532, where EGF has been shown to inhibit IGF-I-dependent degradation of IRS-1 (Zhang et al. 2000).
Gefitinib induces IGF-IR signalling by promoting re-association of IRS-1 with IGF-IR The ability of EGFR to suppress IGF-IR signalling in Tam-R cells, through limiting the availability of IRS-1, has important implications when considering response of these cells to gefitinib challenge. As described by Gee et al. (2006) in this issue, the residual MAPK and AKT signalling observed following anti-hormonal treatment in wild-type MCF-7 breast cancer cells arises from the induction of previously repressed signal transduction genes, such as EGFR. Similarly, the induction of residual IGF-IR-mediated AKT activity www.endocrinology-journals.org
Endocrine-Related Cancer (2006) 13 S89–S97 by gefitinib in our Tam-R cells can now be explained by the removal of this EGFR-mediated suppression of IGF-IR signalling. Therefore, gefitinib treatment of the Tam-R cell line alters the dynamics of the EGFR/IGF-IR/ IRS-1 cross-talk system, blocking EGFR/IRS-1 association, reducing Y896 phosphorylation of IRS-1, promoting re-association of IRS-1 with IGF-IR, which in turn results in increased Y612 IRS-1 phosphorylation and recruitment and activation of the PI-3K/AKT-signalling pathway (Knowlden et al. 2006). Similar results with gefitinib have now also been observed in LNCaP prostate cancer cells, suggesting that EGFR is also the dominant recruiter of IRS-1 in this cell line. The ability of gefitinib to promote IGF-IR signalling in Tam-R cells is further evidenced by the ability of gefitinib to further enhance IGF-II-induced phosphorylation of IRS-1 Y612 and AKT when compared with IGF-II treatment alone (Knowlden et al. 2006). Activation of such a pathway may facilitate the ability of the cells to survive gefitinib challenge in the short-term and long-term and provide a mechanism to drive resistant growth. Indeed, our ‘in house’ tamoxifen–gefitinib-resistant MCF-7 breast cancer cell line, which we have previously demonstrated utilizes the IGF-IR/AKT-signalling pathway to drive resistant growth (Jones et al. 2004), also expresses increased levels of IRS-1 Y612 phosphorylation when compared with the parental Tam-R cells (Knowlden et al. 2006). Therapeutic implications
Since gefitinib-induced IGF-IR signalling appears to play a key role in initially protecting Tam-R cells during the drug-responsive phase and ultimately driving resistant cell growth, targeting IGF-IR in combination with gefitinib could prove effective in improving gefitinib action and potentially delaying or even preventing resistance development. This hypothesis was confirmed by studies assessing the effects of a combination of gefitinib and the selective IGF-IR tyrosine kinase inhibitor ABDP on Tam-R cell signalling and growth. This combination therapy prevents the activation of IGF-IR by IGF-II and blocks the gefitinib-induced enhancement of IRS-1 Y612 and AKT phosphorylation in response to this ligand (Knowlden et al. 2006). Greater inhibition of phosphorylated levels of IRS-1 Y896, EGFR Y1068 and ERK1/2 are also observed in those cells treated with the combination of ABDP and gefitinib compared with gefitinib alone, reflecting the important role played by IGF-IR in facilitating EGFR-signalling activity in Tam-R cells (Knowlden et al. 2005, 2006). This www.endocrinology-journals.org
more effective inhibition of IGF-IR- and EGFRsignalling pathways by combination treatment also translates out into a greater inhibition of cell growth compared with either agent alone (Knowlden et al. 2006). These findings support the work of Camirand et al. (2005) who reported additive or synergistic-inhibitory effects on breast cancer cell growth in cells treated with a combination of the selective IGF-IR tyrosine kinase inhibitor, AG1024, and gefitinib compared with either agent alone. We have also sought to examine the effects of long-term combination therapy targeting EGFR and IGF-IR. At 4–5 months, we have found that Tam-R cells treated with gefitinib alone show evidence of regrowth, with cell numbers doubling over a 2-week time frame, as previously reported (Jones et al. 2005). However, Tam-R cells treated with a combination of gefitinib and ABDP demonstrate a considerable reduction in cell numbers at this same time point (Knowlden et al. 2006). These findings support the previous studies examining combination treatment of Tam-R cells with gefitinib and the IGF-IR tyrosine kinase inhibitor, AG1024 (Nicholson et al. 2004), and indicate that targeting the gefitinibinduced IGF-IR signalling in Tam-R cells may provide a mechanism to prevent cells surviving the initial challenge with this anti-EGFR agent and ultimately block the development of a resistant phenotype.
Microarray analysis reveals novel gefitinib-induced genes The EGFR has the capacity to influence intracellular signalling both at the non-genomic and at the genomic levels. We have clearly established a gefitinib-resistant mechanism that functions at the non-genomic level, through influences on IRS-1 and PI-3K/AKT phosphorylation levels; however, a role for alternative genomic resistance mechanisms to this anti-EGFR agent cannot be ignored. To evaluate the effect of gefitinib on gene transcription, we have turned to the use of Affymetrix microarray analysis. These studies are beginning to reveal the full breadth of growth factor-signalling elements promoted during challenge with gefitinib. One example is the glial-cell line-derived neurotrophic factor (GDNF) family receptor a3 (GFRa3) gene which transcribes a plasma membrane-localized Ret co-receptor present at significant levels in Tam-R cells. Following treatment of Tam-R cells with gefitinib for 10 days, we found this gene to be further increased in expression both at the mRNA and at the protein levels (Burmi et al. 2005). Since GFRa3 has been implicated in Ret-mediated cell survival signalling in other cell types, its induction with gefitinib may allow S93
I R Hutcheson et al.: Gefitinib-induced signalling mechanisms maintenance of residual cell viability and hence acquisition of resistance in the presence of this inhibitor (Sariola & Saarma 2003). In accordance with this concept, we have observed that exogenous addition of the GFRa3 ligand, artemin is able to entirely overcome the growth-inhibitory effects of gefitinib in Tam-R cells (Burmi et al. 2005). Preliminary studies focussed on potential gefitinibinduced tyrosine kinases in Tam-R cells have also revealed a number of signalling genes with potential roles in cell survival and resistance. These include fibroblast growth factor receptors, which have been implicated in the genesis of a wide range of human cancer types (Grose & Dickson 2005). In addition, the induction of the receptor tyrosine kinase c-erbB2, a member of the c-erbB receptor family along with EGFR, has also been observed in response to gefitinib. c-erbB2 is also an anti-hormone-induced gene and has been clearly established as a key promoter of cell survival and proliferative pathways associated with anti-hormone resistance in breast cancer (Knowlden et al. 2003, Nicholson et al. 2004). Interestingly, other anti-hormone-induced tyrosine kinase genes, such as Lyn and the ephrin A4 receptor (see Gee et al. in this issue), have also been identified as gefitinib inducible in Tam-R cells, which may reflect the ability of EGFR signalling to cross-talk and re-activate oestrogen receptor signalling in these cells (Britton et al. 2005). Therefore, gefitinib also acts as an anti-hormonal agent in this cell line. Finally, two interesting tyrosine kinases linked to cell motility and invasion in cancer, namely Met and FAK, have also been identified as gefitinib inducible genes. We have found that our acquired gefitinibresistant TAM/TKI-R cell line demonstrate a four- to eightfold increase in motility and ability to invade through matrigel compared with the parental Tam-R cells (Hiscox et al. 2004). The induction of Met and FAK following acute gefitinib treatment would suggest that the generation of these more aggressive features may be an early event in the gefitinib resistance process.
between EGFR and IGF-IR in tamoxifen-resistant MCF-7 breast and LNCaP prostate cell lines whereby recruitment of IRS-1 by EGFR can limit the availability of IRS-1 to associate with IGF-IR and, as a result, potentially inhibit signalling via this receptor. This suppression of IGF-IR signalling by EGFR can be disrupted by treatment with gefitinib with the resultant association of IRS-1 with IGF-IR leading to reestablishment of IGF-IR signalling, the dominant growth-regulatory mechanism of gefitinib resistance in these cells. Therefore, gefitinib plays an active role in limiting its own efficacy in Tam-R cells by promoting activation of a resistance pathway. These findings clearly demonstrate that, as a consequence of the high degree of cross-talk that exists between growth factorsignalling pathways in cancer cells, we must take into consideration that targeting a single protein in this complex signalling array may have wide-ranging and unexpected effects on a number of signal transduction pathways which may adversely influence the quality and duration of response. Our Affymetrix data are also demonstrating the huge redundancy in signalling pathways regulating the fundamental biological processes of cell survival and proliferation so that alongside this non-genomic mechanism, there is a considerable potential for a range of alternative gefitinib-induced genomic resistance mechanisms, all or some of which may serve to limit gefitinib response. On the positive side, if we can start deciphering these potential inductive effects, in conjunction with the known inhibitory functions of these targeted agents, this knowledge will provides us with the opportunity to design effective strategies to combat such resistance mechanisms and improve response to initial therapy. Indeed, as proof of principle, we have demonstrated that targeting IGF-IR signalling in combination with gefitinib, to anticipate the inductive action of gefitinib on this pathway, generates a more effective inhibition of Tam-R cell signalling activity and growth compared with gefitinib alone and has the potential to prevent resistance development.
Conclusions
Acknowledgements
In conclusion, there is a clear need to understand the resistance mechanisms to targeted therapies, such as the selective EGFR tyrosine kinase inhibitor gefitinib, if we are to improve the effectiveness of these treatments. Here, we identify a novel physical interaction between the EGFR and the adaptor protein IRS-1 in a range of EGFR-positive breast, prostate and lung cancer cell lines. This ability of EGFR to recruit IRS-1 highlights a novel cross-talk mechanism
Many thanks to the members of the Tissue Culture, Gene Discovery, Immunocytochemistry and Cell Signalling units for their contribution to this work.
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Funding This research was generously supported by the Tenovus Organisation. R S Burmi was funded on a studentship by the Breast Cancer Campaign. www.endocrinology-journals.org
Endocrine-Related Cancer (2006) 13 S89–S97 The authors declare the following potential conflicts of interest regarding this research: H E Jones, J M W Gee and R I Nicholson are in receipt of funding from AstraZeneca and R I Nicholson is also a member of an advisory board for AstraZeneca.
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