Intrinsic and acquired resistance to EGFR inhibitors in human cancer therapy
http://www.100md.com
《肿瘤与内分泌》
1 Dipartimento di Endocrinologia Molecolare e Clinica, Università degli Studi di Napoli ‘Federico II’, Naples, Italy
2 Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale ‘F. Magrassi e A. Lanzara’, Seconda Università degli Studi di Napoli, Naples, Italy
This paper was presented at the 1st Tenovus/AstraZeneca Workshop, Cardiff (2005). AstraZeneca has supported the publication of these proceedings.
Abstract
The epidermal growth factor receptor (EGFR) autocrine pathway plays a crucial role in human cancer since it contributes to a number of highly relevant processes in tumor development and progression, including cell proliferation, regulation of apoptotic cell death, angiogenesis and metastatic spread. Among a variety of approaches used to target EGFR signaling, EGFR blocking monoclonal antibodies and small molecular weight EGFR tyrosine kinase compounds have been successfully developed. The results of a large body of preclinical studies and clinical trials suggest that targeting the EGFR could represent a significant contribution to cancer therapy. Both types of agent exert a significant antiproliferative activity when used alone or in combination with conventional antitumor treatments, such as chemotherapy or radiation therapy. Although the advanced clinical development of EGFR blocking drugs demonstrates their efficacy in some human metastatic diseases, such as lung, head and neck and colorectal cancers, the issue of constitutive resistance in a large number of patients and the development of acquired resistance in the responders remains an unexplored subject of investigation. Recent evidence suggests the role of specific activating mutations within the tyrosine kinase domain of EGFR to explain the dramatic responses to small molecule tyrosine kinase inhibitors in a subgroup of lung cancer patients. However, the intrinsic molecular mechanisms of resistance to these drugs are still unclear. This review will focus on the preclinical findings on therapeutic resistance to EGFR targeting agents.
Introduction
In the last decade significant progress has been made in the understanding of the molecular mechanisms which are responsible for human cancer development and progression. A large body of preclinical and clinical findings has highlighted the role of the epidermal growth factor receptor (EGFR) family in tumor formation and maintenance. EGFR is widely expressed by many cell types, including epithelial and mesenchymal lineages (Wells 1999). In human malignancies the incidence of overexpression and/or dysregulation of this receptor is variable. This variability may be partially explained by a lack of standardization in the methodology for detection of EGFR expression in cancer specimens, mainly for immunohistochemistry. Overall, the most common human epithelial cancers generally express the EGFR, although gene amplification is not commonly reported. EGFR is, in fact, frequently overexpressed in human tumors such as head and neck squamous cell carcinoma (HNSCC), glioblastoma, non-small cell lung cancer (NSCLC), and breast, colorectal (CRC), bladder, prostate and ovarian carcinomas (Salomon et al. 1995, Mendelsohn 2001). The type-III mutated variant of the human EGFR, denominated EGFRvIII and characterized by a deletion in the extracellular domain that leads to constitutive activation of its tyrosine kinase (TK) domain, is the most frequently expressed EGFR genetic alteration in some cancers, like glioblastomas (Nishikawa et al. 1994, Moscatello et al. 1995, Kuan et al. 2001).
Overexpression of EGFR correlates with poor prognosis and worse clinical outcome in a large number of malignancies, including NSCLC, bladder, breast and HNSC cancers (Moscatello et al. 1995, Grandis et al. 1998). Nicholson and colleagues (Nicholson et al. 2001) reviewed 200 studies involving more than 20 000 patients to determine the prognostic value of increased EGFR expression for reduced recurrence-free or overall survival rates and found a strong prognostic value for HNSCC, ovarian, bladder, cervical and esophageal cancers, a moderate prognostic value for CRC, breast, gastric and endometrial tumors, and only a weak prognostic value for NSCLC. Moreover, increased receptor content is often associated with an increased production of specific activating ligands, such as transforming growth factor (TGF), by the same tumor cells, leading to receptor activation through an autocrine stimulatory pathway (Rusch et al. 1993, Salomon et al. 1995, Grandis et al. 1998).
Several strategies for EGFR targeting have been proposed, including small molecule tyrosine kinase inhibitors (TKIs) that interfere with receptor phosphorylation, monoclonal antibodies (MAbs) that directly interfere with ligand-receptor binding, antisense oligonucleotides or ribozymes that block mRNA receptor translation in a functioning protein (Yamazaki et al. 1998, Ciardiello et al. 2001a) and, finally, MAbs which serve as carriers of radionuclides, prodrugs or toxins (Azemar et al. 2000). Of these approaches, low-molecular weight TKIs and blocking MAbs are in the most advanced stages of clinical development (Ciardiello & Tortora 2001). MAbs function at the extracellular ligand-binding site of the EGFR, whereas small molecule TKIs function at the intracellular tyrosine kinase domain of the EGFR. The efficacy in cancer treatment of some of these inhibitors is testified by a massive amount of preclinical data and by clinical trials in advanced chemorefractory cancers, including HNSCC, NSCLC and CRC. These data have led to the recent introduction into clinical practice of cetuximab (Erbitux), a chimerized human-murine IgG1 anti-EGFR blocking MAb, which has been approved for irinotecan-refractory, metastatic colorectal cancer, and of two low molecular weight synthetic, selective EGFR-TKIs, gefitinib (Iressa) and erlotinib (Tarceva), which have been licensed for the second and third line treatment, respectively, of advanced chemorefractory NSCLC.
Despite high levels of EGFR expression within the tumor, some patients are clearly refractory to EGFR inhibitor treatment, suggesting that mere EGFR expression is not a reliable predictor of response to therapy and revealing the emerging importance of understanding the molecular mechanisms responsible for cancer cell resistance to such inhibitors. A preclinical study on 60 human cancer cell lines of the United States National Cancer Institute Anticancer Drug Screen has been performed with several EGFR family inhibitors (Bishop et al. 2002) and has revealed that the level of EGFR expression for a specific tumor is less important than the degree of activation of the EGFR-dependent intracellular pathways in predicting the response to EGFR-targeted therapy. In fact, EGFR activation can be affected from several factors, such as receptor homo- or hetero-dimerization with each of the other three members of the EGFR family (ErbB-2, ErbB-3 or ErbB-4), increased expression of different ligands and EGFR mutations (Arteaga 2002). The lack of a simple relationship between the levels of EGFR expression and the degree of its activation renders it difficult to predict the clinical effectiveness of EGFR targeted therapeutics (Arteaga & Baselga 2003) (Table 1). For such reasons, the investigation of the molecular mechanisms which predict for sensitivity or which lead to resistance to EGFR signal transduction inhibitors is an important and clinically relevant field of cancer research.
Mechanisms of resistance to EGFR inhibitors
Cancer cell resistance to EGFR antagonists could be due to several reasons. Human cancer cells carry genetic alterations which enable them to have a dys-regulated proliferation and an intrinsic resistance to anticancer drugs. Furthermore, the genetic instability of these cells could cause the apprearance of cancer cell clones with an acquired resistance following prolonged exposure to EGFR inhibitors (Nowell 1976, Lengauer et al. 1998). On the other hand, host-related mechanisms could also be responsible for intrinsic cancer cell resistance, such as a defective immune system-mediated function, a rapid inactivating metabolism or a poor absorption. For example, impairment of antibody- dependent cell-mediated cytotoxicity (ADCC), an important mechanism of action of several MAbs in vivo, including the anti-EGFR MAb cetuximab and the anti-ErbB-2 MAb trastuzumab (Herceptin) (Clynes et al. 2000), could result in an intrinsic host-related resistance to treatment with these agents. Since EGFR antagonists interfere with the activation of several intracellular pathways that control cell proliferation, survival, apoptosis, metastatic capability, invasion and tumor-induced angiogenesis, it is also clear that several different molecular changes important in EGFR-dependent or -independent cellular signaling pathways could be responsible for the development of resistance to these inhibitors. In the following paragraphs, we will summarize the experimental evidence of the different molecular mechanisms which may be responsible for both constitutive and acquired cancer cell resistance to selective EGFR antagonists (Table 2).
Activation of alternative growth factor receptor signaling pathways
The genetic instability of cancer cells with the contribution of drug-induced selective pressure, confers the ability to switch to alternative survival mechanisms in case of inhibition of crucial pathways which are necessary for cell survival. These escape mechanisms support the rationale for combining multiple anticancer agents with different mechanism of action to obtain better clinical efficacy of an anticancer treatment. One of the potential molecular pathways which is used by cancer cells as an alternative survival system to overcome the blockage of EGFR function induced by specific EGFR inhibitors is represented by the activation of other tyrosine kinase receptor systems which are not EGFR-related, such as the insulin-like growth factor (IGF) family of ligands and receptors. The association between IGF-I and human tumors is supported by the clinical evidence that higher levels of circulating IGF-I are associated with increased risk of several cancers (Chan et al. 1998, Holly 1998, Yu et al. 2000) and by the experimental evidence that IGF-I signaling pathways are altered in cancer cells (Resnicoff & Baserga 1998, Nickerson et al. 2001). IGF-I receptor (IGF-IR) activation stimulates signaling pathways involved in mitogenesis, cell survival and resistance to apoptotic cell death (LeRoith et al. 1995). IGF-IR capability to interfere with the growth inhibitory action of the anti-ErbB-2 MAb trastuzumab has been recently demonstrated (Lu et al. 2001). Trastuzumab is able to inhibit the proliferation of ErbB-2 (Baselga et al. 1998, Pietras et al. 1998) and it is used alone or in combination with different chemotherapeutic drugs in the treatment of breast cancer patients with ErbB-2 overexpressing tumors. However, only 15 to 35% of breast cancer patients with ErbB-2 overxpressing tumors respond to therapy with trastuzumab and the development of resistance is a common clinical problem. Trastuzumab is able to inhibit the growth of ErbB-2 overexpressing and IGF-IR expressing human MCF-7/HER2 and SKBR3 breast cancer cells only when the IGF-IR-driven signals are blocked. In fact, in low-serum conditions the growth of breast cancer cells is strongly reduced by the addition of trastuzumab to the culture media, but, in the presence of 10% fetal bovine serum or in presence of exogenous IGF-I, trastuzumab has no effect on the proliferation of these breast cancer cell lines. This lack of antiproliferative effect of trastuzumab appears more evident when cancer cells with low expression of IGF-IR, like SKBR3, are genetically altered to overexpress IGF-IR and are cultured in the presence of IGF-I to activate a positive autocrine loop. The addition of IGF-I-binding protein-3, which decreases IGF-IR signaling, results in restoration of trastuzumab-induced growth inhibition in these cells (Lu et al. 2001). In SKBR3 cells transfected with IGF-IR, IGF-I treatment antagonizes the trastuzumab-induced increase in the level of the cyclin-dependent kinase (CDK) inhibitor p27KIP1, with a consequently decreased association of p27KIP1 with CDK2, restoration of CDK2 activity and, therefore, attenuation of cell cycle arrest in the G1 phase, all of which are induced by trastuzumab treatment in trastuzumab-sensitive breast cancer cells (Lu et al. 2004). Moreover, the ability of IGF-I to confer resistance to trastuzumab seems to also involve the targeting of p27KIP1 to the ubiquitin/ proteasome degradation machinery in a phosphoinositide 3-kinase (PI3K)-dependent pathway (Lu et al. 2004).
The relationship between increased IGF-IR activity and the efficacy of small molecule EGFR-TKIs, such as the quinazoline derivative AG1478, has been investigated in human glioblastoma multiforme (GBM) cell lines (Chakravarti et al. 2002). In this study, despite equivalent EGFR levels and a similar reduction in EGFR signaling, as measured by EGFR tyrosine phosphorylation, following treatment with AG1478 the antiproliferative activity of EGFR blockade was different in various GBM cell lines. In fact, AG1478 was able to induce apoptosis and to reduce invasive potential only in sensitive GBM cell lines. The resistant phenotype correlated in GBM cancer cells with enhanced IGF-IR levels following AG1478 administration and with sustained signaling through the PI3K-akt pathway. In fact, the small molecule EGFR TKI AG1478 failed to inhibit the phosphorylation and activation of the antiapoptotic effector Akt GBM resistant cells, whereas it induced a significant reduction in PI3K and akt activition in GBM sensitive cells. In this study, combined targeting of IGF-IR and EGFR by using AG1478 and AG1024, a specific IGF-IR inhibitor, greatly enhanced apoptosis and reduced the invasive potential of GBM resistant cells (Chakravarti et al. 2002). More recently, a correlation between IGF-IR activation and acquired resistance to EGFR blockade was also demonstrated for breast and prostate cancer cell lines (Jones et al. 2004). Continuous exposure to gefitinib for up to 6 months of the EGFR-positive, gefitinib-sensitive, tamoxifen-resistant, MCF- 7 breast cancer cells (TAM-R) caused the occurrence of a stable, gefitinib-resistant subline (TAM/TKI-R). As compared with parental TAM-R cells, the TAM/ TKI-R cells showed no detectable basal phosphorylated EGFR activity, but elevated levels of IGF-IR, protein kinase C (PKC) and Akt. Treatment of the gefitinib-resistant TAM/TKI-R cell line with the specific IGF-IR inhibitor AG1024 resulted in significant growth inhibition and in reduced migratory capacity. Similarly, a gefitinib-resistant variant of the EGFR-positive, gefitinib-sensitive, androgen-independent human prostate cancer cell line DU145 has been generated by selective drug pressure following long-term exposure to gefitinib (DU145/TKI-R cells). Also in this case, the EGFR-resistant phenotype was associated with an increased signaling via the IGF-IR pathway (Jones et al. 2004).
Activation of EGFR-independent, tumor-induced angiogenesis
Tumor-induced angiogenesis is well known as a key player in sustaining local tumor growth, invasion and metastatic spread. In cancer cells the EGFR autocrine pathway controls, in part, the production of several proangiogenic growth factors, including vascular endothelial growth factor (VEGF) (Goldman et al. 1993, Gille et al. 1997) and basic fibroblast growth factor (bFGF) (Ciardiello et al. 2001b). The link between EGFR and VEGF signaling is also testified by the major anticancer effect due to one of EGFR by selective anti-EGFR agents of tumor-induced, VEGF-mediated angiogenesis (Ciardiello et al. 1996, Petit et al. 1997). In cancer cells, altered control of angiogenesis could be a mechanism responsible for resistance to EGFR inhibitors in vivo, as it has been shown in preclinical models with anti-EGFR blocking MAbs. Human A431 squamous cell carcinoma xenografts, which possess an amplified EGFR gene with high expression of EGFR protein, are extremely sensitive to the antitumor effects of EGFR targeting agents. However, long term treatment of A431 tumor xenografts bearing severe combined immunodeficiency (SCID) mice with each of three different EGFR blocking MAbs (mR3, hR3 or cetuximab) results in the appearance of A431 tumor xenografts which are resistant to these drugs (Viloria-Petit et al. 2001). Six A431-derived, in vivo resistant cell lines were isolated and characterized. When cultured in vitro, these cell lines, retained a high level of EGFR expression, exhibited an unaltered growth rate and were sensitive to the antiproliferative effects of anti-EGFR MAbs as compared with parental A431 cells. However, when reinjected in vivo in immunodefiecient mice all EGFR-inhibitor resistant cell lines displayed a resistant behavior. Five of six resistant A431-derived clones expressed increased levels of VEGF, which in turn increased tumor angiogenesis in vivo. Further, exogenous expression of VEGF gene transfection of A431 sensitive, parental cells renders these cells significantly resistant to anti-EGFR MAbs when injected in nude mice, demonstrating the causal role of VEGF dysregulated overexpression in the acquired resistance (Viloria-Petit et al. 2001). Therefore, it is possible that in tumor models with increased VEGF expression, which is independent from EGFR signaling, resistance to EGFR antagonists could be the consequence of their inability to down-regulate VEGF production in cancer cells. Moreover, all of the A431 cell variants resistant in vivo to EGFR inhibitors carry other molecular alterations that could contribute to the resistant phenotype, such as overexpression of cyclin D1 and Bcl-XL (Viloria-Petit & Kerbel 2004): the first one facilitates cell cycle progression from G1 to S phase, the second one functions as a repressor of cell death. Both cyclin D1 and Bcl-XL when overexpressed are associated with improved survival capabilities and are positively regulated by EGFR signaling. Downregulation of these molecules by EGFRs of ErbB-2 antagonists seems to play a crucial role in their growth-inhibitory and proapoptotic effects (Yakes et al. 2002). High expression levels of Bcl-XL in the A431-resistant variants are independent of EGFR signaling, which could represent a possible involvement of this antiapoptotic pathway in the resistant phenotype (Viloria-Petit & Kerbel 2004).
The impact of VEGF signaling in cancer cell resistance in EGFR-expressing tumors has been investigated in our laboratory through the generation of human GEO colon cancer cell lines resistant to either small molecule EGFR-TKIs, such as gefitinib, or to anti-EGFR MAbs, such as cetuximab (Ciardiello et al. 2004). GEO cell resistant sublines have been established from GEO xenografts growing in nude mice which were treated chronically with one of these two EGFR inhibitors. After an initial tumor regression, almost all the treated animals experienced tumor regrowth at the site of inoculation after a variable latency period despite continuous therapy with the EGFR inhibitor. In contrast, continuous treatment of mice bearing GEO xenografts with ZD6474, a small molecule VEGF .k-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also has activity against the EGFR tyrosine kinase, resulted in efficient tumor growth inhibition for the entire duration of treatment (up to five months). Interestingly, sequential ZD6474 treatment of GEO tumor xenografts following cetuximab or gefitinib resulted in the block of tumor growth, in contrast to re-treatment with either selective anti- EGFR agent. Cell lines derived from such resistant tumors (named GEO-C225-RES and GEO-ZD1839- RES for resistance to cetuximab or to gefitinib, respectively) have been characterized. Protein expression analysis revealed no major changes in the expression of cell membrane EGFR or of the EGFR ligand TGF, of Bcl-2, Bcl-XL, p53, MDM2, Akt, activated Akt, and MAPK. However, both GEOC225- RES and GEO-ZD1839-RES cells exhibited a 5- to 10-fold increase in activated phospho-MAPK, in the expression of cyclooxygenase-2 (COX-2) and of VEGF as compared with parental EGFR-inhibitor sensitive GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either cetuximab or gefitinib but was efficiently inhibited by ZD6474. These data confirm the previous reported experimental evidence by which acquired resistance to EGFR antagonists might arise from enhanced VEGF expression rather than loss in the expression or a functional alteration of EGFR signaling. However, the balance between VEGF and EGFR receptors signaling in causing and sustaining resistance to EGFR inhibitors appears to be also dependent on tumor cell type. In this respect, a gefitinib-resistant lung adenocarcinoma cell line (PC-9/ ZD) has been recently characterized with a similar profile of lack of sensitivity to both gefitinib and ZD6474 in vivo as compared with gefitinib- and ZD6474- sensitive PC-9 parental cells, suggesting the activation of other EGFR-independent biochemical pathways which are responsible for the resistant phenotype (Taguchi et al. 2004).
Independent activation of intracellular molecular effectors which function downstream to the EGFR
The plethora of effectors, kinases, protein adapters and phosphatases which are involved in cancer cell growth explains the complex network of molecular interactions that could activate signaling pathways independently of EGFR activity. Aberrant activation of intracellular signaling elements such as PI3K represents one of the most common reported mechanisms by which resistance to EGFR inhibitors might arise. Increased and EGFR-independent activity of PI3K could result from direct gene amplification, activating mutations of p85 subunits, overexpression of downstream effectors such as Akt or inactivating mutations or loss of function of regulators such as PTEN, a phosphatase that acts as a negative regulator of PI3K. These alterations are common events during cancer formation and progression and could possibly result in constitutive activation of oncogenic signals through Akt, MAPK or both. Amplification of PI3K with concomitant overexpression and increased enzymatic activity has been observed in ovarian and cervical cancer (Shayesteh et al. 1999, Ma et al. 2000) and has been suggested as an early molecular change in ovarian carcinogenesis. Similarly, amplification of the type 2 isozyme of Akt (Akt2) has been observed in ovarian and pancreatic carcinomas (Cheng et al. 1992, 1996) and correlates with metastatic advanced disease. Loss of PTEN expression, as a consequence of gene mutations and/or deletions, as well as of gene silencing, occurs with variable frequency in advanced cancers including glioblastoma multiforme, melanoma, endometrial, breast, ovarian, renal cell, and thyroid cancer, and a small subset of NSCLC (Ali et al. 1999, Vivanco & Sawyers et al. 2002). Reconstitution of PTEN expression in PTEN-null cells has been shown to repress Akt and inhibit tumor growth via induction of apoptosis or repression of cell proliferation (Li & Sun 1998, Lu et al. 1999). The absence of a functional PTEN protein in cancer cells suggests it could be responsible for the resistance of EGFR family expressing cancer cells to specific inhibitors. In fact, whereas the growth of the human squamous carcinoma cell line A431 is markedly inhibited by gefitinib treatment, human breast cancer MDA-468 cells, which carry a deletion and a frame-shift mutation at codon 70 of the PTEN gene (Lu et al. 1999) with loss of functional PTEN protein, are relatively resistant to gefitinib treatment (Bianco et al. 2003). Although gefitinib treatment blocks EGFR autophosphorylation and coupling with p85 in both A431 and MDA-468 EGFR overexpressing cell lines, the basal activity of the PI3K target Akt is suppressed only in A431 cells, implying that Akt activity in MDA-468 cells is independent of EGFR signals and possesses a high threshold that is unresponsive to EGFR inhibition alone. The introduction of a functional PTEN gene with PTEN protein expression in MDA-468 cells results in restoring gefitinib-induced Akt inhibition, in relocalization of the Akt target Forkhead factor FKHRL to the cell nucleus and in increased FKHRL1-dependent transcriptional activity. Moreover, gefitinib treatment causes a significant inhibition of cell growth and apoptosis in PTEN-reconstituted MDA-468 cells similar to those observed in A431 cells (Bianco et al. 2003). These effects are also observed with other two EGFR inhibitors such as erlotinib and cetuximab. Therefore, since one of the main functions of PTEN is to counteract PI3K in the regulation of phosphoinositide signaling, a consequence of PTEN loss is unbalanced PI3K signaling and constitutive activation of the PI3K/Akt pathway, which is responsible of acquired resistance to EGFR antagonists. In PTEN-deleted tumor cells, a more balanced phosphoinositide signaling may also be achieved using pharmacologic inhibitors of PI3K kinase (She et al. 2003). Low and non-effective doses of LY294002, a PI3K inhibitor, are able to sensitize MDA-468 cells to gefitinib treatment, by reducing basal Akt activity and reestablishing EGFR-stimulated Akt signaling. The differential sensitivity to EGFR inhibitors as a function of PTEN status is not limited to A431 and MDA-468 cells. H157 human NSCLC cells lacks PTEN protein (Forgacs et al. 1998) and display resistance to gefitinib as compared with H1355 human NSCLC cells, which possess similar levels of EGFR and a wild type PTEN, and are growth-inhibited upon gefitinib treatment (Bianco et al. 2003). In addition, in ErbB-2 overexpressing breast cancer cells, trastuzumab treatment results in PTEN activation through increased PTEN membrane localization and phosphatase activity and inhibition of src association with ErbB-2 (Nagata et al. 2004). Reducing PTEN activity by specific antisense oligonucleotides seems to confer resistance to trastuzumab, both in vitro and in vivo. Patients with PTEN-deficient breast cancers have significantly poorer responses to trastuzumab-based therapy than those with normal PTEN (Nagata et al. 2004). Taken together these results suggest that PTEN inactivation is a predictor for resistance to EGFR-family antagonists.
Effects of EGFR gene mutations and of the loss of the target
It is possible that loss of EGFR expression or altered function due to EGFR gene mutations can be a mechanism by which tumor cells became resistant to EGFR antagonists. Acquired mutations within the Bcr-Abl gene have been shown as responsible for the development of clinical resistance to the Bcr-Abl kinase inhibitor imatinib mesylate (Gleevec; STI-571) in patients affected by chronic myeloid leukaemia (CML) (Roumiantsev et al. 2002, Roche-Lestienne et al. 2002). A C to T single nucleotide change in the Abl kinase domain, which substitutes Threonine 315 with isoleucine, is the most frequently reported mutation. A corresponding mutation in the EGFR tyrosine kinase domain, that replaces Threonine 766 with methionine, dramatically reduces the sensitivity to the growth inhibitory effects of the small molecule EGFR-TKI PD153035 (Blencke et al. 2003).
Furthermore, GBM cell lines expressing the mutated variant EGFR-vIII appear to be relatively resistant to gefitinib since higher doses and longer exposure to gefitinib are necessary to signifcantly decrease EGFRvIII phosphorylation. Cell cycle analysis shows that nascent DNA synthesis in EGFR-expressing cells is inhibited in a dose-dependent manner by gefitinib, whereas it is unaffected in EGFRvIII-expressing cells. The protective activity of EGFRvIII may be due in part to phosphorylation of Akt, which is inhibited in EGFR expressing cells after treatment with gefitinib, but is unaffected in cells expressing EGFRvIII (Learn et al. 2004).
However, a large body of clinical data has been recently provided on the predictive role of EGFR gene mutations and enhanced sensitivity to EGFR inhibitors. Recent landmark publications have shown that specific somatic mutations in the kinase domain of EGFR in some some patients with advanced and chemorefractory NSCLC are associated with dramatic and long lasting clinical responses to the tyrosine kinase inhibitor gefitinib. These mutations in the EGFR seem to play a significant role in determining the sensitivity of tumor cells to small molecule EGFR-TKIs such as gefitinib and erlotinib by altering the tridimensional conformation and activity of the receptor. Paez and colleagues (Paez et al. 2004) searched for somatic genetic alterations in a set of 119 primary NSCLC tumors by sequencing the EGFR gene. Somatic mutations (missense mutations G719S and L858R and Del-1 deletion) were found in the EGFR kinase domain and correlated strikingly with specific patient characteristics since mutations were more frequent in adenocarcinomas (21%) than in other NSCLCs (2%), more frequent in women (20%) than in man (9%), and more frequent in Japanese patients. The highest proportion of EGFR mutations were observed in Japanese women with adenocarcinoma (57%). More interestingly, the patient characteristics that correlate with the presence of EGFR mutations were the same that correlate with clinical response to gefitinib. H3255 cells, a lung adenocarcinoma cell line with the EGFR L858R mutation, demonstrates high sensitivity to growth inhibition induced by gefitinib, whereas three other NSCLC cell lines (H1781, H1666 and H441 cells) expressing wild type EGFR are resistant to these EGFR antagonist. In H3255 cells, treatment with gefitinib completely inhibits EGFR autophosphorylation, as well as blocking the phosphorylation of known downstream targets of EGFR such as ERK1/2 and Akt. In contrast, the other three cell lines show comparable levels of inhibition of EGFR phosphorylation only when gefitinib is present at concentrations roughly 100 times higher.
Lynch and colleagues (Lynch et al. 2004) sequenced the entire coding region of the gene using PCR amplification of individual exons. Heterozygous mutations were observed in eight of nine patients defined as responders to gefitinib, all of which were clustered within the tyrosine kinase domain of EGFR. Four tumors had in-frame deletions within exon 19, another four tumors had amino acid substitutions within exon 21 of the tyrosine kinase domain. By comparison, no EGFR mutations were observed in seven patients with non small cell lung cancer who had no response to gefitinib. To study the functional properties of the EGFR encoded by these mutated genes, EGFR with the L747–P753 in-frame deletion and EGFR with the L858R missense mutation were expressed in Cos-7 cells. In the absence of serum and of activating growth factors, neither wild-type nor mutant EGFR demonstrated autophosphorylation. However, EGF treatment caused a three- to four-fold increase in EGFR phosphorylation in both mutant EGFRs as compared with the activation of the wild-type EGFR. Moreover, the two mutant receptors had a continued activation for up to three hours. Remarkably, both mutant receptors were more sensitive than the wildtype receptor to inhibition by gefitinib. Since all the mutations are clustered near the ATP cleft of the tyrosine kinase domain, where they flank amino acids shown in crystallographic studies to mediate binding of 4-anilinoquinazoline compounds, such as gefitinib (Stamos et al. 2002), it is possible that the mutations result in repositioning of these critical residues, stabilizing their interaction with both ATP and its competitive inhibitor gefitinib. Such a mechanism could explain both the increased receptor activation after ligand binding and the enhanced inhibition induced by gefitinib. The effects of mutated variants of EGFR has also been investigated in other NSCLC cell lines, such as H1650, that carries the in-frame deletion E746-A750, or H1975, with missense mutation L858R, or NCI358, H1666 and H1734 that have wildtype EGFR (Sordella et al. 2004). Cell lines harbouring EGFR mutations displayed a selective activation of anti-apoptotic signals through Akt and signal transduction and activator of transcription (STAT) 5, which promote cell survival but have no effect on Erk, which induces proliferation. EGF-induced autophosphorylation of tyrosine992 (Y992) and tyrosine1068 (Y1068) was markedly elevated in the two cancer cell lines harboring EGFR gene mutations, with a concomitant increase in phosphorylation of Akt and STAT5 but not of Erk. The two lung cancer cell lines harboring EGFR mutations exhibited an increased proliferative activity as relative to lung cancer cells expressing wild-type EGFR when maintained in the presence of EGF in low serum concentrations. However, the proliferation rate and cell density at confluence were comparable at normal serum concentrations. Moreover, NSCLC cells expressing mutant EGFRs underwent extensive apoptosis after small interfering RNA (siRNA)–mediated knockdown of the mutant EGFR or treatment with pharmacological inhibitors of Akt and STAT signaling and were relatively resistant to apoptosis induced by conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. Thus, these mutant EGFRs selectively transduce survival signals on which NSCLCs become dependent and it is possible that NSCLCs expressing only wild-type receptors do not display a similar dependence on EGFR activation and this could account for the relative gefitinib-insensitivity of human NSCLC that express wild-type EGFR.
Somatic mutations in the EGFR TK domain have also been associated with sensitivity to erlotinib (Pao et al. 2004). Five of seven NSCLC tumor specimens from patients which were sensitive to erlotinib treatment had analogous somatic mutations (in-frame deletions within exon 19 or point mutations within exon 21), as opposed to no mutations found in 10 erlotinib-refractory tumors. Most EGFR mutation-positive tumors were adenocarcinomas from patients who have never smoked.
The issue of EGFR mutations in sensitivity/resistance to EGFR inhibitors has been addressed also for non-selective EGFR antagonists, such as ZD6474 (Arao et al. 2004). A strong correlation has been noted in several cancer cells lines in vitro between the IC50 values of gefitinib and ZD6474; conversely no correlation was observed between the sensitivity to ZD6474 and the level of EGFR or VEGFR expression. The NSCLC cell line PC-9 was hypersensitive to gefitinib and ZD6474, and a small (15-bp) in-frame deletion of the ATP-binding site (exon 19) in the EGFR TK domain was detected (delE746-A750–type deletion). The involvement of this EGFR mutation in the cellular sensitivity to ZD6474 has been confirmed by transfection of HEK293 cells with the EGFR mutated construct. These cells exhibited a 60-fold higher sensitivity to ZD6474 as compared with cells expressing wild-type EGFR. ZD6474 inhibited the phosphorylation of the mutant EGFR by 10-fold as compared with cells with wild-type EGFR. The correlation between ‘gain of function’, somatic EGFR mutations and sensitivity to small molecule EGFR-TKIs has been documented in NSCLC cell lines and patients. However, similar mutations have not been found or appear to be very rare in other cancer types, including breast, glioblastomas, CRC and HNSCC (Barber et al. 2004, Lynch et al. 2004, Lee et al. 2005).
Other mechanisms of cancer cell resistance to EGFR inhibitors
Cyclin D1 and p27KIP1 are downstream effectors involved in EGFR-dependent intracellular mitogenic signaling (Busse et al. 2001, Lenferink et al. 2001, Hulit et al. 2002), which are commonly deregulated in various cancers (Jares et al. 1994, Bova et al. 1999, Okami et al. 1999). The relationship between deregulated Cyclin D1 expression and sensitivity to gefitinib has been investigated to determine whether this frequently occurring oncogenic change could affect the cellular response to this EGFR-TKI (Kalish et al. 2004). In a panel of six EGFR-overexpressing HNSCC lines analyzed, three of them which were carrying Cyclin D1 gene amplification and/or protein overexpression displayed resistance to gefitinib. SCC9 HNSCC cells transfected with a Cyclin D1 expression vector had a sustained proliferation with a high Sphase fraction when treated with gefitinib, whereas empty vector control clones and the parental SCC 9 cells were significantly growth-inhibited with a marked reduction in S-phase. The resistance of Cyclin D1- overexpressing clones and Cyclin D1-amplified cell lines was associated with maintenance of Cyclin D1 protein expression after gefitinib treatment.
Recently, proteome pattern studies have been performed to understand the cellular signals underlying resistance to MAbs against EGFR. To define proteins involved in EGFR-triggered growth regulation and potential resistance mechanisms, the proteome profile of human colorectal cancer cell lines with high expression of functional EGFR but different sensitivity to cetuximab has been characterized (Skvortsov et al. 2004). Cetuximab treatment resulted in a complete saturation of EGFR in Caco-2 and HRT-18 CRC cell lines. However, whereas Caco-2 cells showed inhibition of proliferation, growth of HRT-18 cells was not suppressed. Using two-dimensional electrophoresis gels and subsequent mass spectrometry, the authors identified 14 proteins differentially expressed in the two cell lines. Among these proteins, fatty acid binding protein and heat shock protein 27 might contribute to the resistance of HRT-18 CRC cells to cetuximab treatment. These data demonstrate that proteome-based investigations could be an useful tool to better understand the complex protein interactions involved in EGFR signaling and in resistance to EGFR inhibitors.
Sensitivity to EGFR antagonists could be also related to genetic differences among individuals. The hypothesis that genetic variations in the EGFR gene could explain the resistant phenotype has been tested (Amador et al. 2004). In fact, several studies have revealed that polymorphic variations in genes encoding drug targets affect the response and toxicity to therapeutic agents (Arranz et al. 1995, Yoshida et al. 1995, Benetos et al. 1996, van Essen et al. 1996, Henrion et al. 1998, Lima et al. 1999). The EGFR gene contains a highly polymorphic sequence in intron 1, which consists of a variable number of CA dinucleotide repeats ranging from 9 to 21 (Chrysogelos 1993). This sequence has been shown to affect the efficiency of gene transcription such that subjects or cell lines with a greater number of CA repeats have lower levels of EGFR mRNA and protein expression (Gebhardt et al. 1999, Buerger et al. 2000). HNSCC lines with lower numbers of CA dinucleotides in the CA single sequence repeat (CA-SSR) of the intron 1 had a higher expression of EGFR and were more sensitive to the growth inhibitory effects of erlotinib. Phenotypic modification by silencing EGFR mRNA expression in HN029 cells sensitive to erlotinib induced resistance to this drug. The analysis of clinical specimens obtained from tumor and paired peripheral blood cells from 30 patients with advanced HNSCC revealed an equivalent number of CA dinucleotides between paired samples of the same individual, supporting the notion that this region is not commonly somatically mutated in the process of tumor development.
For some common anticancer agents, one possible mechanism of resistance is increased drug efflux resulting from the activity of membrane-associated pumps, such as P-glycoprotein (P-gp), the product of the multidrug resistance gene mdr-1. Since many small molecule inhibitors of tyrosine kinase have a neutral and hydrophobic nature, they could be substrates for P-gp or similar-acting efflux pumps. Intracellular accumulation of CI-1033, a small molecule EGFR-TKI, seems to be dependent on the breast cancer resistance protein (BCRP), a recently cloned ATP binding cassette transporter. In MDA-MB-231 breast cancer cells, transfection of BCRP resulted in a decrease in CI-1033 accumulation, compared with cells transfected with empty vector s(Erlichman et al. 2001). This observation suggests that CI-1033 is itself a substrate for BCRP, which in turn could possibly regulate the efflux of other specific EGFR inhibitors.
Conclusions
It is now well established that EGFR signaling is important to both normal development and to neoplastic transformation and that EGFR inhibition represents a valid anti-cancer approach. Different EGFR molecular antagonists have been evaluated in a clinical setting and some of these drugs have demonstrated dramatic clinical efficacy in a subset of NSCLC patients (in particular, gefitinib and erlotinib), as well as in HNSCC and colorectal cancer patients (in particular, cetuximab). However, constitutive and acquired resistance to EGFR inhibitors represent a major and unsolved clinical problem in treating cancer patients with EGFR expressing tumors. Since the enhanced and uncontrolled activation of key intracellular signal transduction pathways, the use of alternative growth factor receptor signals, the presence of EGFR gene alterations might constitute a common series of mechanisms which cause resistance to EGFR blockade, it is reasonable that only novel combined molecularly targeted therapeutic approaches aimed to block both the EGFR and these mechanisms may represent an effective anticancer treatment for a larger group of patients whose tumors have a functional EGFR pathway.
Acknowledgements
The research program in the laboratory of the authors was performed with a research grant from AIRC (Associazione Italiana per la Ricerca sul Cancro), Italy.
References
Ali IU, Schriml LM & Dean M 1999 Mutational spectra of PTEN/MMAC1 gene a tumor suppressor with lipid phosphatase activity. Journal of the National Cancer Institute 17 1922–1932.
Amador ML, Oppenheimer D, Perea S, Maitra A, Cusati G, Iacobuzio-Donahue C, Baker SD, Ashfaq R, Takimoto C, Forastiere A & Hidalgo M 2004 An epidermal growth factor receptor intron 1 polymorphism mediates response to epidermal growth factor receptor inhibitors. Cancer Research 15 9139–9143.
Arao T, Fukumoto H, Takeda M, Tamura T, Saijo N & Nishio K 2004 Small in-frame deletion in the epidermal growth factor receptor as a target for ZD6474. Cancer Research 15 9101–9104.
Arranz M, Collier D, Sodhi M, Ball D, Roberts G, Price J, Sham P & Kerwin R 1995 Association between clozapine response and allelic variation in 5-HT2A receptor gene. Lancet 346 281–282.
Arteaga CL 2002 Epidermal growth factor receptor dependence in human tumors more than just expression Oncologist 7 (Suppl 4) 31–39.
Arteaga CL & Baselga J 2003 Clinical trial design and end points for epidermal growth factor receptor-targeted therapies implications for drug development and practice. Clinical Cancer Research 9 1579–1589.
Azemar M, Schmidt M, Arlt F, Kennel P, Brandt B, Papadimitriou A, Groner B & Wels W 2000 Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapid tumor regression in vivo. International Journal of Cancer 15 269–275.
Barber TD, Vogelstein B, Kinzler KW & Velculescu VE 2004 Somatic mutations in colorectal cancer and glioblastomas. New England Journal of Medicine 351 2883.
Baselga J, Norton L, Albanell J, Kim YM & Mendelsohn J 1998 Recombinant humanized anti- HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Research 58 2825–2831.
Benetos A, Cambien F, Gautier S, Ricard S, Safar M, Laurent S, Lacolley P, Poirier O, Topouchian J & Asmar R 1996 Influence of the angiotensin II type 1 receptor gene polymorphism on the effects of perindopril and nitrendipine on arterial stiffness in hypertensive individuals. Hypertension 28 1081–1084.
Bianco R, Shin I, Ritter CA, Yakes FM, Basso A, Rosen N, Tsurutani J, Dennis PA, Mills GB & Arteaga CL 2003 Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors. Oncogene 8 2812–2822.
Bishop PC, Myers T, Robey R, Fry DW, Liu ET, Blagosklonny MV & Bates SE 2002 Differential sensitivity of cancer cells to inhibitors of the epidermal growth factor receptor family. Oncogene 3 119–127.
Blencke S, Ullrich A & Daub H 2003 Mutation of threonine 766 in the epidermal growth factor receptor reveals a hotspot for resistance formation against selective tyrosine kinase inhibitors. The Journal of Biological Chemistry 25 15435–15440.
Bova RJ, Quinn DI, Nankervis JS, Cole IE, Sheridan BF, Jensen MJ, Morgan GJ, Hughes CJ & Sutherland RL 1999 Cyclin D1 and p16INK4A expression predict reduced survival in carcinoma of the anterior tongue. Clinical Cancer Research 5 2810–2819.
Buerger H, Gebhardt F, Schmidt H, Beckmann A, Hutmacher K, Simon R, Lelle R, Boecker W & Brandt B 2000 Length and loss of heterozygosity of an intron 1 polymorphic sequence of egfr is related to cytogenetic alterations and epithelial growth factor receptor expression. Cancer Research 60 854–857.
Busse D, Doughty RS & Arteaga CL 2001 HER-2/neu (erbB- 2) and the cell cycle. Seminars in Oncology 27 (Suppl 11) 3–8.
Chakravarti A, Loeffler JS & Dyson NJ 2002 Insulin-like growth factor receptor I mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation of phosphoinositide 3-kinase signaling. Cancer Research 1 200–207.
Chan JM, Stampfer MJ, Giovannucci E, Gann PH, Ma J, Wilkinson P, Hennekens CH & Pollak M 1998 Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science 23 563–566.
Cheng JQ, Godwin AK, Bellacosa A, Taguchi T, Franke TF, Hamilton TC, Tsichlis PN & Testa JR 1992 AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. PNAS 1 9267–9271.
Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA, Watson DK & Testa JR 1996 Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. PNAS 16 3636–3641.
Chrysogelos SA 1993 Chromatin structure of the EGFR gene suggests a role for intron 1 sequences in its regulation in breast cancer cells. Nucleic Acids Research 21 5736–5741.
Ciardiello F, Damiano V, Bianco R, Bianco C, Fontanini G, De Laurentiis M, De Placido S, Mendelsohn J, Bianco AR & Tortora G 1996 Antitumor activity of combined blockade of epidermal growth factor receptor and protein kinase A. Journal of the National Cancer Institute 4 1770–1776.
Ciardiello F & Tortora G 2001 A novel approach in the treatment of cancer targeting the epidermal growth factor receptor. Clinical Cancer Research 7 2958–2970.
Ciardiello F, Caputo R, Troiani T, Borriello G, Kandimalla ER, Agrawal S, Mendelsohn J, Bianco AR & Tortora G 2001a Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis, and cooperate with cytotoxic drugs in human cancer cell lines. International Journal of Cancer 15 172–178.
Ciardiello F, Caputo R, Bianco R, Damiano V, Fontanini G, Cuccato S, De Placido S, Bianco AR & Tortora G 2001b Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clinical Cancer Research 7 1459–1465.
Ciardiello F, Bianco R, Caputo R, Caputo R, Damiano V, Troiani T, Melisi D, De Vita F, De Placido S, Bianco AR & Tortora G 2004 Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy. Clinical Cancer Research 15 784–793.
Clynes RA, Towers TL, Presta LG & Ravetch JV 2000 Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nature Medicine 6 443–446.
Erlichman C, Boerner SA, Hallgren CG, Spieker R, Wang XY, James CD, Scheffer GL, Maliepaard M, Ross DD, Bible KC & Kaufmann SH 2001 The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast cancer resistance protein-mediated drug efflux. Cancer Research 15 739–748.
Forgacs E, Biesterveld EJ, Sekido Y, Fong K, Muneer S, Wistuba II, Milchgrub S, Brezinschek R, Virmani A, Gazdar AF & Minna JD 1998 Mutation analysis of the PTEN/MMAC1 gene in lung cancer. Oncogene 24 1557–1565.
Gebhardt F, Zanker KS & Brandt B 1999 Modulation of epidermal growth factor receptor gene transcription by a polymorphic dinucleotide repeat in intron 1. The Journal of Biological Chemistry 274 13176–13180.
Gille J, Swerlick RA & Caughman SW 1997 Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation. EMBO J 17 750–759.
Goldman CK, Kim J, Wong WL, King V, Brock T & Gillespie GY 1993 Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells a model of glioblastoma multiforme pathophysiology. Molecular Biology of the Cell 4 121–133.
Grandis JR, Melhem MF, Gooding WE, Day R, Holst VA, Wagener MM, Drenning SD & Tweardy DJ 1998 Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival. Journal of the National Cancer Institute 90 824–832.
Henrion D, Amant C, Benessiano J, Philip I, Plantefeve G, Chatel D, Hwas U, Desmont JM, Durand G, Amouyel P & Levy BI 1998 Angiotensin II type 1 receptor gene polymorphism is associated with an increased vascular reactivity in the human mammary artery in vitro. Journal of Vascular Research 35 356–362.
Holly JM 1998 Insulin-like growth factor-I and new opportunities for cancer prevention. Lancet 351 1373–1375.
Huang SF, Liu HP, Li LH, Ku YC, Fu YN, Tsai HY, Chen YT, Lin YF, Chang WC, Kuo HP, Wu YC, Chen YR & Tsai SF 2004 High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clinical Cancer Research 15 8195–8203.
Hulit J, Lee RJ, Russell RG & Pestell RG 2002 ErbB-2-induced mammary tumor growth: the role of cyclin D1 and p27Kip1. Biochemical Pharmacology 64 827–836.
Jares P, Fernandez PL, Campo E, Nadal A, Bosch F, Aiza G, Nayach I, Traserra J & Cardesa A 1994 PRAD-1/cyclin D1 gene amplification correlates with messenger RNA overexpression and tumor progression in human laryngeal carcinomas. Cancer Research 1 4813–4817.
Jones HE, Goddard L, Gee JM, Hiscox S, Rubini M, Barrow D, Knowlden JM, Williams S, Wakeling AE & Nicholson RI 2004 Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells. Endocrine-Related Cancer 11 793–814.
Kalish LH, Kwong RA, Cole IE, Gallagher RM, Sutherland RL & Musgrove EA 2004 Deregulated cyclin D1 expression is associated with decreased efficacy of the selective epidermal growth factor receptor tyrosine kinase inhibitor gefitinib in head and neck squamous cell carcinoma cell lines. Clinical Cancer Research 15 7764–7774.
Kuan CT, Wikstrand CJ & Bigner DD 2001 EGF mutant receptor vIII as a molecular target in cancer therapy. Endocrine-Related Cancer 8 83–96.
Learn CA, Hartzell TL, Wikstrand CJ, Archer GE, Rich JN, Friedman AH, Friedman HS, Bigner DD & Sampson JH 2004 Resistance to tyrosine kinase inhibition by mutant epidermal growth factor receptor variant III contributes to the neoplastic phenotype of glioblastoma multiforme. Clinical Cancer Research 1 3216–3224.
Lee JW, Soung YH, Kim SY, Park WS, Nam SW, Lee JY, Yoo NJ & Lee SH 2005 Absence of EGFR mutations in the kinase domain in common human cancers beside non-small cell lung cancer. International Journal of Cancer 113 510–511.
Lenferink AE, Busse D, Flanagan WM, Yakes FM & Arteaga CL 2001 ErbB2/neu kinase modulates cellular p27(Kip1) and cyclin D1 through multiple signaling pathways. Cancer Research 1 6583–6591.
Lengauer C, Kinzler KW & Vogelstein B 1998 Genetic instabilities in human cancers. Nature 396 643–649.
LeRoith D, Werner H, Beitner-Johnson D & Roberts CT Jr 1995 Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocrine Reviews 16 143–163.
Li B, Chang CM, Yuan M, McKenna WG & Shu HK 2003 Resistance to small molecule inhibitors of epidermal growth factor receptor in malignant gliomas. Cancer Research 1 7443–7450.
Li DM & Sun H 1998 PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells. PNAS 22 15406–15411.
Lima JJ, Thomason DB, Mohamed MH, Eberle LV, Self TH & Johnson JA 1999 Impact of genetic polymorphisms of the beta2-adrenergic receptor on albuterol bronchodilator pharmacodynamics. Clinical Pharmacology and Therapeutics 65 519–525.
Lu Y, Lin YZ, LaPushin R, Cuevas B, Fang X, Yu SX, Davies MA, Khan H, Furui T, Mao M, Zinner R, Hung MC, Steck P, Siminovitch K & Mills GB 1999 The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene 25 7034–45.
Lu Y, Zi X & Pollak M 2004 Molecular mechanisms underlying IGF-I-induced attenuation of the growth-inhibitory activity of trastuzumab (Herceptin) on SKBR3 breast cancer cells. International Journal of Cancer 20 334–341.
Lu Y, Zi X, Zhao Y, Mascarenhas D & Pollak M 2001 Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). Journal of the National Cancer Institute 19 1852–1857.
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J & Haber DA 2004 Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. New England Journal of Medicine 20 2129–2139.
Ma YY, Wei SJ, Lin YC, Lung JC, Chang TC, Whang-Peng J, Liu JM, Yang DM, Yang WK & Shen CY 2000 PIK3CA as an oncogene in cervical cancer. Oncogene 25 2739–2544.
Mendelsohn J 2001 The epidermal growth factor receptor as a target for cancer therapy. Endorine-Related Cancer 8 3–9.
Moscatello DK, Holgado-Madruga M, Godwin AK, Ramirez G, Gunn G, Zoltick PW, Biegel JA, Hayes RL & Wong AJ 1995 Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Research 1 5536–5539.
Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, Klos KS, Li P, Monia BP, Nguyen NT, Hortobagyi GN, Hung MC & Yu D 2004 PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6 117–127.
Nicholson RI, Gee JM & Harper ME 2001 EGFR and cancer prognosis. European Journal of Cancer 37 (Suppl 4) 9–15.
Nickerson T, Chang F, Lorimer D, Smeekens SP, Sawyers CL & Pollak M 2001 In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR). Cancer Research 61 6276–6280.
Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK & Huang HJ 1994 A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. PNAS 2 7727–7731.
Nowell PC 1976 The clonal evolution of tumor cell populations. Science 194 23–28.
Okami K, Reed AL, Cairns P, Koch WM, Westra WH, Wehage S, Jen J & Sidransky D 1999 Cyclin D1 amplification is independent of p16 inactivation in head and neck squamous cell carcinoma. Oncogene 10 3541–3545.
Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE & Meyerson M 2004 EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 4 1497–500.
Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, Singh B, Heelan R, Rusch V, Fulton L, Mardis E, Kupfer D, Wilson R, Kris M & Varmus H 2004 EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. PNAS 7 13306–13311.
Petit AM, Rak J, Hung MC, Rockwell P, Goldstein N, Fendly B & Kerbel RS 1997 Neutralizing antibodies against EGF and ErbB-2/neu receptor tyrosine kinases down-regulate VEGF production by tumor cells in vitro and in vivo Angiogenic implications for signal transduction therapy of solid tumors. American Journal of Pathology 151 1523–1530.
Pietras RJ, Pegram MD, Finn RS, Maneval DA & Slamon DJ 1998 Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs. Oncogene 17 2235–2249.
Resnicoff M & Baserga R 1998 The role of the insulin-like growth factor I receptor in transformation and apoptosis. Annals of the New York Academy of Sciences 842 76–81.
Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, Lai JL, Philippe N, Facon T, Fenaux P & Preudhomme C 2002 Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 100 1014–1018.
Roumiantsev S, Shah NP, Gorre ME, Nicoll J, Brasher BB, Sawyers CL & Van Etten RA 2002 Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. PNAS 6 10700–10705.
Rusch V, Baselga J, Cordon-Cardo C, Orazem J, Zaman M, Hoda S, McIntosh J, Kurie J & Dmitrovsky E 1993 Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Research 15 (Suppl 10) 2379–2385.
Salomon DS, Brandt R, Ciardiello F & Normanno N 1995 Epidermal growth factor-related peptides and their receptors in human malignancies. Critical Reviews in Oncology/Hematology 19 183–232.
Shayesteh L, Lu Y, Kuo WL, Baldocchi R, Godfrey T, Collins C, Pinkel D, Powell B, Mills GB & Gray JW 1999 PIK3CA is implicated as an oncogene in ovarian cancer. Nature Genetics 21 99–102.
She QB, Solit D, Basso A & Moasser MM 2003 Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'-kinase/Akt pathway signaling. Clinical Cancer Research 1 4340–4346.
Skvortsov S, Sarg B, Loeffler-Ragg J, Skvortsova I, Lindner H, Werner Ott H, Lukas P, Illmensee K & Zwierzina H 2004 Different proteome pattern of epidermal growth factor receptor-positive colorectal cancer cell lines that are responsive and nonresponsive to C225 antibody treatment. Molecular Cancer Therapeutics 3 1551–1558.
Sordella R, Bell DW, Haber DA & Settleman J 2004 Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 20 1163–1167.
Stamos J, Sliwkowski MX & Eigenbrot C 2002 Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. The Journal of Biological Chemistry 29 46265–46272.
Taguchi F, Koh Y, Koizumi F, Tamura T, Saijo N & Nishio K 2004 Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib (‘Iressa’)-sensitive and resistant xenograft models. Cancer Science 1 984–989.
van Essen GG, Rensma PL, de Zeeuw D, Sluiter WJ, Scheffer H, Apperloo AJ & de Jong PE 1996 Association between angiotensinconverting-enzyme gene polymorphism and failure of renoprotective therapy. Lancet 347 94–95.
Viloria-Petit A, Crombet T, Jothy S, Hicklin D, Bohlen P, Schlaeppi JM, Rak J & Kerbel RS 2001 Acquired resistance to the antitumor effect of epidermal growth factor receptorblocking antibodies in vivo A role for altered tumor angiogenesis. Cancer Research 61 5090–5101.
Viloria-Petit AM & Kerbel RS 2004 Acquired resistance to EGFR inhibitors mechanisms and prevention strategies. International Journal of Radiation Oncology Biology Physics 1 914–926.
Vivanco I & Sawyers CL 2002 The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nature Reviews Cancer 2 489–501.
Wells A 1999 EGF receptor. The International Journal of Biochemistry and Cell Biology 31 637–643.
Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S & Arteaga CL 2002 Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Research 15 4132–4141.
Yamazaki H, Kijima H, Ohnishi Y, Abe Y, Oshika Y, Tsuchida T, Tokunaga T, Tsugu A, Ueyama Y, Tamaoki N & Nakamura M 1998 Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression. Journal of the National Cancer Institute 15 581–587.
Yoshida H, Mitarai T, Kawamura T, Kitajima T, Miyazaki Y, Nagasawa R, Kawaguchi Y, Kubo H, Ichikawa I & Sakai O 1995 Role of the deletion of polymorphism of the angiotensin converting enzyme gene in the progression and therapeutic responsiveness of IgA nephropathy. The Journal of Clinical Investigation 96 2162–2169.
Yu H & Rohan T 2000 Role of the insulin-like growth factor family in cancer development and progression. Journal of the National Cancer Institute 92 1472–1489.(Roberto Bianco, Teresa Tr)
2 Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale ‘F. Magrassi e A. Lanzara’, Seconda Università degli Studi di Napoli, Naples, Italy
This paper was presented at the 1st Tenovus/AstraZeneca Workshop, Cardiff (2005). AstraZeneca has supported the publication of these proceedings.
Abstract
The epidermal growth factor receptor (EGFR) autocrine pathway plays a crucial role in human cancer since it contributes to a number of highly relevant processes in tumor development and progression, including cell proliferation, regulation of apoptotic cell death, angiogenesis and metastatic spread. Among a variety of approaches used to target EGFR signaling, EGFR blocking monoclonal antibodies and small molecular weight EGFR tyrosine kinase compounds have been successfully developed. The results of a large body of preclinical studies and clinical trials suggest that targeting the EGFR could represent a significant contribution to cancer therapy. Both types of agent exert a significant antiproliferative activity when used alone or in combination with conventional antitumor treatments, such as chemotherapy or radiation therapy. Although the advanced clinical development of EGFR blocking drugs demonstrates their efficacy in some human metastatic diseases, such as lung, head and neck and colorectal cancers, the issue of constitutive resistance in a large number of patients and the development of acquired resistance in the responders remains an unexplored subject of investigation. Recent evidence suggests the role of specific activating mutations within the tyrosine kinase domain of EGFR to explain the dramatic responses to small molecule tyrosine kinase inhibitors in a subgroup of lung cancer patients. However, the intrinsic molecular mechanisms of resistance to these drugs are still unclear. This review will focus on the preclinical findings on therapeutic resistance to EGFR targeting agents.
Introduction
In the last decade significant progress has been made in the understanding of the molecular mechanisms which are responsible for human cancer development and progression. A large body of preclinical and clinical findings has highlighted the role of the epidermal growth factor receptor (EGFR) family in tumor formation and maintenance. EGFR is widely expressed by many cell types, including epithelial and mesenchymal lineages (Wells 1999). In human malignancies the incidence of overexpression and/or dysregulation of this receptor is variable. This variability may be partially explained by a lack of standardization in the methodology for detection of EGFR expression in cancer specimens, mainly for immunohistochemistry. Overall, the most common human epithelial cancers generally express the EGFR, although gene amplification is not commonly reported. EGFR is, in fact, frequently overexpressed in human tumors such as head and neck squamous cell carcinoma (HNSCC), glioblastoma, non-small cell lung cancer (NSCLC), and breast, colorectal (CRC), bladder, prostate and ovarian carcinomas (Salomon et al. 1995, Mendelsohn 2001). The type-III mutated variant of the human EGFR, denominated EGFRvIII and characterized by a deletion in the extracellular domain that leads to constitutive activation of its tyrosine kinase (TK) domain, is the most frequently expressed EGFR genetic alteration in some cancers, like glioblastomas (Nishikawa et al. 1994, Moscatello et al. 1995, Kuan et al. 2001).
Overexpression of EGFR correlates with poor prognosis and worse clinical outcome in a large number of malignancies, including NSCLC, bladder, breast and HNSC cancers (Moscatello et al. 1995, Grandis et al. 1998). Nicholson and colleagues (Nicholson et al. 2001) reviewed 200 studies involving more than 20 000 patients to determine the prognostic value of increased EGFR expression for reduced recurrence-free or overall survival rates and found a strong prognostic value for HNSCC, ovarian, bladder, cervical and esophageal cancers, a moderate prognostic value for CRC, breast, gastric and endometrial tumors, and only a weak prognostic value for NSCLC. Moreover, increased receptor content is often associated with an increased production of specific activating ligands, such as transforming growth factor (TGF), by the same tumor cells, leading to receptor activation through an autocrine stimulatory pathway (Rusch et al. 1993, Salomon et al. 1995, Grandis et al. 1998).
Several strategies for EGFR targeting have been proposed, including small molecule tyrosine kinase inhibitors (TKIs) that interfere with receptor phosphorylation, monoclonal antibodies (MAbs) that directly interfere with ligand-receptor binding, antisense oligonucleotides or ribozymes that block mRNA receptor translation in a functioning protein (Yamazaki et al. 1998, Ciardiello et al. 2001a) and, finally, MAbs which serve as carriers of radionuclides, prodrugs or toxins (Azemar et al. 2000). Of these approaches, low-molecular weight TKIs and blocking MAbs are in the most advanced stages of clinical development (Ciardiello & Tortora 2001). MAbs function at the extracellular ligand-binding site of the EGFR, whereas small molecule TKIs function at the intracellular tyrosine kinase domain of the EGFR. The efficacy in cancer treatment of some of these inhibitors is testified by a massive amount of preclinical data and by clinical trials in advanced chemorefractory cancers, including HNSCC, NSCLC and CRC. These data have led to the recent introduction into clinical practice of cetuximab (Erbitux), a chimerized human-murine IgG1 anti-EGFR blocking MAb, which has been approved for irinotecan-refractory, metastatic colorectal cancer, and of two low molecular weight synthetic, selective EGFR-TKIs, gefitinib (Iressa) and erlotinib (Tarceva), which have been licensed for the second and third line treatment, respectively, of advanced chemorefractory NSCLC.
Despite high levels of EGFR expression within the tumor, some patients are clearly refractory to EGFR inhibitor treatment, suggesting that mere EGFR expression is not a reliable predictor of response to therapy and revealing the emerging importance of understanding the molecular mechanisms responsible for cancer cell resistance to such inhibitors. A preclinical study on 60 human cancer cell lines of the United States National Cancer Institute Anticancer Drug Screen has been performed with several EGFR family inhibitors (Bishop et al. 2002) and has revealed that the level of EGFR expression for a specific tumor is less important than the degree of activation of the EGFR-dependent intracellular pathways in predicting the response to EGFR-targeted therapy. In fact, EGFR activation can be affected from several factors, such as receptor homo- or hetero-dimerization with each of the other three members of the EGFR family (ErbB-2, ErbB-3 or ErbB-4), increased expression of different ligands and EGFR mutations (Arteaga 2002). The lack of a simple relationship between the levels of EGFR expression and the degree of its activation renders it difficult to predict the clinical effectiveness of EGFR targeted therapeutics (Arteaga & Baselga 2003) (Table 1). For such reasons, the investigation of the molecular mechanisms which predict for sensitivity or which lead to resistance to EGFR signal transduction inhibitors is an important and clinically relevant field of cancer research.
Mechanisms of resistance to EGFR inhibitors
Cancer cell resistance to EGFR antagonists could be due to several reasons. Human cancer cells carry genetic alterations which enable them to have a dys-regulated proliferation and an intrinsic resistance to anticancer drugs. Furthermore, the genetic instability of these cells could cause the apprearance of cancer cell clones with an acquired resistance following prolonged exposure to EGFR inhibitors (Nowell 1976, Lengauer et al. 1998). On the other hand, host-related mechanisms could also be responsible for intrinsic cancer cell resistance, such as a defective immune system-mediated function, a rapid inactivating metabolism or a poor absorption. For example, impairment of antibody- dependent cell-mediated cytotoxicity (ADCC), an important mechanism of action of several MAbs in vivo, including the anti-EGFR MAb cetuximab and the anti-ErbB-2 MAb trastuzumab (Herceptin) (Clynes et al. 2000), could result in an intrinsic host-related resistance to treatment with these agents. Since EGFR antagonists interfere with the activation of several intracellular pathways that control cell proliferation, survival, apoptosis, metastatic capability, invasion and tumor-induced angiogenesis, it is also clear that several different molecular changes important in EGFR-dependent or -independent cellular signaling pathways could be responsible for the development of resistance to these inhibitors. In the following paragraphs, we will summarize the experimental evidence of the different molecular mechanisms which may be responsible for both constitutive and acquired cancer cell resistance to selective EGFR antagonists (Table 2).
Activation of alternative growth factor receptor signaling pathways
The genetic instability of cancer cells with the contribution of drug-induced selective pressure, confers the ability to switch to alternative survival mechanisms in case of inhibition of crucial pathways which are necessary for cell survival. These escape mechanisms support the rationale for combining multiple anticancer agents with different mechanism of action to obtain better clinical efficacy of an anticancer treatment. One of the potential molecular pathways which is used by cancer cells as an alternative survival system to overcome the blockage of EGFR function induced by specific EGFR inhibitors is represented by the activation of other tyrosine kinase receptor systems which are not EGFR-related, such as the insulin-like growth factor (IGF) family of ligands and receptors. The association between IGF-I and human tumors is supported by the clinical evidence that higher levels of circulating IGF-I are associated with increased risk of several cancers (Chan et al. 1998, Holly 1998, Yu et al. 2000) and by the experimental evidence that IGF-I signaling pathways are altered in cancer cells (Resnicoff & Baserga 1998, Nickerson et al. 2001). IGF-I receptor (IGF-IR) activation stimulates signaling pathways involved in mitogenesis, cell survival and resistance to apoptotic cell death (LeRoith et al. 1995). IGF-IR capability to interfere with the growth inhibitory action of the anti-ErbB-2 MAb trastuzumab has been recently demonstrated (Lu et al. 2001). Trastuzumab is able to inhibit the proliferation of ErbB-2 (Baselga et al. 1998, Pietras et al. 1998) and it is used alone or in combination with different chemotherapeutic drugs in the treatment of breast cancer patients with ErbB-2 overexpressing tumors. However, only 15 to 35% of breast cancer patients with ErbB-2 overxpressing tumors respond to therapy with trastuzumab and the development of resistance is a common clinical problem. Trastuzumab is able to inhibit the growth of ErbB-2 overexpressing and IGF-IR expressing human MCF-7/HER2 and SKBR3 breast cancer cells only when the IGF-IR-driven signals are blocked. In fact, in low-serum conditions the growth of breast cancer cells is strongly reduced by the addition of trastuzumab to the culture media, but, in the presence of 10% fetal bovine serum or in presence of exogenous IGF-I, trastuzumab has no effect on the proliferation of these breast cancer cell lines. This lack of antiproliferative effect of trastuzumab appears more evident when cancer cells with low expression of IGF-IR, like SKBR3, are genetically altered to overexpress IGF-IR and are cultured in the presence of IGF-I to activate a positive autocrine loop. The addition of IGF-I-binding protein-3, which decreases IGF-IR signaling, results in restoration of trastuzumab-induced growth inhibition in these cells (Lu et al. 2001). In SKBR3 cells transfected with IGF-IR, IGF-I treatment antagonizes the trastuzumab-induced increase in the level of the cyclin-dependent kinase (CDK) inhibitor p27KIP1, with a consequently decreased association of p27KIP1 with CDK2, restoration of CDK2 activity and, therefore, attenuation of cell cycle arrest in the G1 phase, all of which are induced by trastuzumab treatment in trastuzumab-sensitive breast cancer cells (Lu et al. 2004). Moreover, the ability of IGF-I to confer resistance to trastuzumab seems to also involve the targeting of p27KIP1 to the ubiquitin/ proteasome degradation machinery in a phosphoinositide 3-kinase (PI3K)-dependent pathway (Lu et al. 2004).
The relationship between increased IGF-IR activity and the efficacy of small molecule EGFR-TKIs, such as the quinazoline derivative AG1478, has been investigated in human glioblastoma multiforme (GBM) cell lines (Chakravarti et al. 2002). In this study, despite equivalent EGFR levels and a similar reduction in EGFR signaling, as measured by EGFR tyrosine phosphorylation, following treatment with AG1478 the antiproliferative activity of EGFR blockade was different in various GBM cell lines. In fact, AG1478 was able to induce apoptosis and to reduce invasive potential only in sensitive GBM cell lines. The resistant phenotype correlated in GBM cancer cells with enhanced IGF-IR levels following AG1478 administration and with sustained signaling through the PI3K-akt pathway. In fact, the small molecule EGFR TKI AG1478 failed to inhibit the phosphorylation and activation of the antiapoptotic effector Akt GBM resistant cells, whereas it induced a significant reduction in PI3K and akt activition in GBM sensitive cells. In this study, combined targeting of IGF-IR and EGFR by using AG1478 and AG1024, a specific IGF-IR inhibitor, greatly enhanced apoptosis and reduced the invasive potential of GBM resistant cells (Chakravarti et al. 2002). More recently, a correlation between IGF-IR activation and acquired resistance to EGFR blockade was also demonstrated for breast and prostate cancer cell lines (Jones et al. 2004). Continuous exposure to gefitinib for up to 6 months of the EGFR-positive, gefitinib-sensitive, tamoxifen-resistant, MCF- 7 breast cancer cells (TAM-R) caused the occurrence of a stable, gefitinib-resistant subline (TAM/TKI-R). As compared with parental TAM-R cells, the TAM/ TKI-R cells showed no detectable basal phosphorylated EGFR activity, but elevated levels of IGF-IR, protein kinase C (PKC) and Akt. Treatment of the gefitinib-resistant TAM/TKI-R cell line with the specific IGF-IR inhibitor AG1024 resulted in significant growth inhibition and in reduced migratory capacity. Similarly, a gefitinib-resistant variant of the EGFR-positive, gefitinib-sensitive, androgen-independent human prostate cancer cell line DU145 has been generated by selective drug pressure following long-term exposure to gefitinib (DU145/TKI-R cells). Also in this case, the EGFR-resistant phenotype was associated with an increased signaling via the IGF-IR pathway (Jones et al. 2004).
Activation of EGFR-independent, tumor-induced angiogenesis
Tumor-induced angiogenesis is well known as a key player in sustaining local tumor growth, invasion and metastatic spread. In cancer cells the EGFR autocrine pathway controls, in part, the production of several proangiogenic growth factors, including vascular endothelial growth factor (VEGF) (Goldman et al. 1993, Gille et al. 1997) and basic fibroblast growth factor (bFGF) (Ciardiello et al. 2001b). The link between EGFR and VEGF signaling is also testified by the major anticancer effect due to one of EGFR by selective anti-EGFR agents of tumor-induced, VEGF-mediated angiogenesis (Ciardiello et al. 1996, Petit et al. 1997). In cancer cells, altered control of angiogenesis could be a mechanism responsible for resistance to EGFR inhibitors in vivo, as it has been shown in preclinical models with anti-EGFR blocking MAbs. Human A431 squamous cell carcinoma xenografts, which possess an amplified EGFR gene with high expression of EGFR protein, are extremely sensitive to the antitumor effects of EGFR targeting agents. However, long term treatment of A431 tumor xenografts bearing severe combined immunodeficiency (SCID) mice with each of three different EGFR blocking MAbs (mR3, hR3 or cetuximab) results in the appearance of A431 tumor xenografts which are resistant to these drugs (Viloria-Petit et al. 2001). Six A431-derived, in vivo resistant cell lines were isolated and characterized. When cultured in vitro, these cell lines, retained a high level of EGFR expression, exhibited an unaltered growth rate and were sensitive to the antiproliferative effects of anti-EGFR MAbs as compared with parental A431 cells. However, when reinjected in vivo in immunodefiecient mice all EGFR-inhibitor resistant cell lines displayed a resistant behavior. Five of six resistant A431-derived clones expressed increased levels of VEGF, which in turn increased tumor angiogenesis in vivo. Further, exogenous expression of VEGF gene transfection of A431 sensitive, parental cells renders these cells significantly resistant to anti-EGFR MAbs when injected in nude mice, demonstrating the causal role of VEGF dysregulated overexpression in the acquired resistance (Viloria-Petit et al. 2001). Therefore, it is possible that in tumor models with increased VEGF expression, which is independent from EGFR signaling, resistance to EGFR antagonists could be the consequence of their inability to down-regulate VEGF production in cancer cells. Moreover, all of the A431 cell variants resistant in vivo to EGFR inhibitors carry other molecular alterations that could contribute to the resistant phenotype, such as overexpression of cyclin D1 and Bcl-XL (Viloria-Petit & Kerbel 2004): the first one facilitates cell cycle progression from G1 to S phase, the second one functions as a repressor of cell death. Both cyclin D1 and Bcl-XL when overexpressed are associated with improved survival capabilities and are positively regulated by EGFR signaling. Downregulation of these molecules by EGFRs of ErbB-2 antagonists seems to play a crucial role in their growth-inhibitory and proapoptotic effects (Yakes et al. 2002). High expression levels of Bcl-XL in the A431-resistant variants are independent of EGFR signaling, which could represent a possible involvement of this antiapoptotic pathway in the resistant phenotype (Viloria-Petit & Kerbel 2004).
The impact of VEGF signaling in cancer cell resistance in EGFR-expressing tumors has been investigated in our laboratory through the generation of human GEO colon cancer cell lines resistant to either small molecule EGFR-TKIs, such as gefitinib, or to anti-EGFR MAbs, such as cetuximab (Ciardiello et al. 2004). GEO cell resistant sublines have been established from GEO xenografts growing in nude mice which were treated chronically with one of these two EGFR inhibitors. After an initial tumor regression, almost all the treated animals experienced tumor regrowth at the site of inoculation after a variable latency period despite continuous therapy with the EGFR inhibitor. In contrast, continuous treatment of mice bearing GEO xenografts with ZD6474, a small molecule VEGF .k-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also has activity against the EGFR tyrosine kinase, resulted in efficient tumor growth inhibition for the entire duration of treatment (up to five months). Interestingly, sequential ZD6474 treatment of GEO tumor xenografts following cetuximab or gefitinib resulted in the block of tumor growth, in contrast to re-treatment with either selective anti- EGFR agent. Cell lines derived from such resistant tumors (named GEO-C225-RES and GEO-ZD1839- RES for resistance to cetuximab or to gefitinib, respectively) have been characterized. Protein expression analysis revealed no major changes in the expression of cell membrane EGFR or of the EGFR ligand TGF, of Bcl-2, Bcl-XL, p53, MDM2, Akt, activated Akt, and MAPK. However, both GEOC225- RES and GEO-ZD1839-RES cells exhibited a 5- to 10-fold increase in activated phospho-MAPK, in the expression of cyclooxygenase-2 (COX-2) and of VEGF as compared with parental EGFR-inhibitor sensitive GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either cetuximab or gefitinib but was efficiently inhibited by ZD6474. These data confirm the previous reported experimental evidence by which acquired resistance to EGFR antagonists might arise from enhanced VEGF expression rather than loss in the expression or a functional alteration of EGFR signaling. However, the balance between VEGF and EGFR receptors signaling in causing and sustaining resistance to EGFR inhibitors appears to be also dependent on tumor cell type. In this respect, a gefitinib-resistant lung adenocarcinoma cell line (PC-9/ ZD) has been recently characterized with a similar profile of lack of sensitivity to both gefitinib and ZD6474 in vivo as compared with gefitinib- and ZD6474- sensitive PC-9 parental cells, suggesting the activation of other EGFR-independent biochemical pathways which are responsible for the resistant phenotype (Taguchi et al. 2004).
Independent activation of intracellular molecular effectors which function downstream to the EGFR
The plethora of effectors, kinases, protein adapters and phosphatases which are involved in cancer cell growth explains the complex network of molecular interactions that could activate signaling pathways independently of EGFR activity. Aberrant activation of intracellular signaling elements such as PI3K represents one of the most common reported mechanisms by which resistance to EGFR inhibitors might arise. Increased and EGFR-independent activity of PI3K could result from direct gene amplification, activating mutations of p85 subunits, overexpression of downstream effectors such as Akt or inactivating mutations or loss of function of regulators such as PTEN, a phosphatase that acts as a negative regulator of PI3K. These alterations are common events during cancer formation and progression and could possibly result in constitutive activation of oncogenic signals through Akt, MAPK or both. Amplification of PI3K with concomitant overexpression and increased enzymatic activity has been observed in ovarian and cervical cancer (Shayesteh et al. 1999, Ma et al. 2000) and has been suggested as an early molecular change in ovarian carcinogenesis. Similarly, amplification of the type 2 isozyme of Akt (Akt2) has been observed in ovarian and pancreatic carcinomas (Cheng et al. 1992, 1996) and correlates with metastatic advanced disease. Loss of PTEN expression, as a consequence of gene mutations and/or deletions, as well as of gene silencing, occurs with variable frequency in advanced cancers including glioblastoma multiforme, melanoma, endometrial, breast, ovarian, renal cell, and thyroid cancer, and a small subset of NSCLC (Ali et al. 1999, Vivanco & Sawyers et al. 2002). Reconstitution of PTEN expression in PTEN-null cells has been shown to repress Akt and inhibit tumor growth via induction of apoptosis or repression of cell proliferation (Li & Sun 1998, Lu et al. 1999). The absence of a functional PTEN protein in cancer cells suggests it could be responsible for the resistance of EGFR family expressing cancer cells to specific inhibitors. In fact, whereas the growth of the human squamous carcinoma cell line A431 is markedly inhibited by gefitinib treatment, human breast cancer MDA-468 cells, which carry a deletion and a frame-shift mutation at codon 70 of the PTEN gene (Lu et al. 1999) with loss of functional PTEN protein, are relatively resistant to gefitinib treatment (Bianco et al. 2003). Although gefitinib treatment blocks EGFR autophosphorylation and coupling with p85 in both A431 and MDA-468 EGFR overexpressing cell lines, the basal activity of the PI3K target Akt is suppressed only in A431 cells, implying that Akt activity in MDA-468 cells is independent of EGFR signals and possesses a high threshold that is unresponsive to EGFR inhibition alone. The introduction of a functional PTEN gene with PTEN protein expression in MDA-468 cells results in restoring gefitinib-induced Akt inhibition, in relocalization of the Akt target Forkhead factor FKHRL to the cell nucleus and in increased FKHRL1-dependent transcriptional activity. Moreover, gefitinib treatment causes a significant inhibition of cell growth and apoptosis in PTEN-reconstituted MDA-468 cells similar to those observed in A431 cells (Bianco et al. 2003). These effects are also observed with other two EGFR inhibitors such as erlotinib and cetuximab. Therefore, since one of the main functions of PTEN is to counteract PI3K in the regulation of phosphoinositide signaling, a consequence of PTEN loss is unbalanced PI3K signaling and constitutive activation of the PI3K/Akt pathway, which is responsible of acquired resistance to EGFR antagonists. In PTEN-deleted tumor cells, a more balanced phosphoinositide signaling may also be achieved using pharmacologic inhibitors of PI3K kinase (She et al. 2003). Low and non-effective doses of LY294002, a PI3K inhibitor, are able to sensitize MDA-468 cells to gefitinib treatment, by reducing basal Akt activity and reestablishing EGFR-stimulated Akt signaling. The differential sensitivity to EGFR inhibitors as a function of PTEN status is not limited to A431 and MDA-468 cells. H157 human NSCLC cells lacks PTEN protein (Forgacs et al. 1998) and display resistance to gefitinib as compared with H1355 human NSCLC cells, which possess similar levels of EGFR and a wild type PTEN, and are growth-inhibited upon gefitinib treatment (Bianco et al. 2003). In addition, in ErbB-2 overexpressing breast cancer cells, trastuzumab treatment results in PTEN activation through increased PTEN membrane localization and phosphatase activity and inhibition of src association with ErbB-2 (Nagata et al. 2004). Reducing PTEN activity by specific antisense oligonucleotides seems to confer resistance to trastuzumab, both in vitro and in vivo. Patients with PTEN-deficient breast cancers have significantly poorer responses to trastuzumab-based therapy than those with normal PTEN (Nagata et al. 2004). Taken together these results suggest that PTEN inactivation is a predictor for resistance to EGFR-family antagonists.
Effects of EGFR gene mutations and of the loss of the target
It is possible that loss of EGFR expression or altered function due to EGFR gene mutations can be a mechanism by which tumor cells became resistant to EGFR antagonists. Acquired mutations within the Bcr-Abl gene have been shown as responsible for the development of clinical resistance to the Bcr-Abl kinase inhibitor imatinib mesylate (Gleevec; STI-571) in patients affected by chronic myeloid leukaemia (CML) (Roumiantsev et al. 2002, Roche-Lestienne et al. 2002). A C to T single nucleotide change in the Abl kinase domain, which substitutes Threonine 315 with isoleucine, is the most frequently reported mutation. A corresponding mutation in the EGFR tyrosine kinase domain, that replaces Threonine 766 with methionine, dramatically reduces the sensitivity to the growth inhibitory effects of the small molecule EGFR-TKI PD153035 (Blencke et al. 2003).
Furthermore, GBM cell lines expressing the mutated variant EGFR-vIII appear to be relatively resistant to gefitinib since higher doses and longer exposure to gefitinib are necessary to signifcantly decrease EGFRvIII phosphorylation. Cell cycle analysis shows that nascent DNA synthesis in EGFR-expressing cells is inhibited in a dose-dependent manner by gefitinib, whereas it is unaffected in EGFRvIII-expressing cells. The protective activity of EGFRvIII may be due in part to phosphorylation of Akt, which is inhibited in EGFR expressing cells after treatment with gefitinib, but is unaffected in cells expressing EGFRvIII (Learn et al. 2004).
However, a large body of clinical data has been recently provided on the predictive role of EGFR gene mutations and enhanced sensitivity to EGFR inhibitors. Recent landmark publications have shown that specific somatic mutations in the kinase domain of EGFR in some some patients with advanced and chemorefractory NSCLC are associated with dramatic and long lasting clinical responses to the tyrosine kinase inhibitor gefitinib. These mutations in the EGFR seem to play a significant role in determining the sensitivity of tumor cells to small molecule EGFR-TKIs such as gefitinib and erlotinib by altering the tridimensional conformation and activity of the receptor. Paez and colleagues (Paez et al. 2004) searched for somatic genetic alterations in a set of 119 primary NSCLC tumors by sequencing the EGFR gene. Somatic mutations (missense mutations G719S and L858R and Del-1 deletion) were found in the EGFR kinase domain and correlated strikingly with specific patient characteristics since mutations were more frequent in adenocarcinomas (21%) than in other NSCLCs (2%), more frequent in women (20%) than in man (9%), and more frequent in Japanese patients. The highest proportion of EGFR mutations were observed in Japanese women with adenocarcinoma (57%). More interestingly, the patient characteristics that correlate with the presence of EGFR mutations were the same that correlate with clinical response to gefitinib. H3255 cells, a lung adenocarcinoma cell line with the EGFR L858R mutation, demonstrates high sensitivity to growth inhibition induced by gefitinib, whereas three other NSCLC cell lines (H1781, H1666 and H441 cells) expressing wild type EGFR are resistant to these EGFR antagonist. In H3255 cells, treatment with gefitinib completely inhibits EGFR autophosphorylation, as well as blocking the phosphorylation of known downstream targets of EGFR such as ERK1/2 and Akt. In contrast, the other three cell lines show comparable levels of inhibition of EGFR phosphorylation only when gefitinib is present at concentrations roughly 100 times higher.
Lynch and colleagues (Lynch et al. 2004) sequenced the entire coding region of the gene using PCR amplification of individual exons. Heterozygous mutations were observed in eight of nine patients defined as responders to gefitinib, all of which were clustered within the tyrosine kinase domain of EGFR. Four tumors had in-frame deletions within exon 19, another four tumors had amino acid substitutions within exon 21 of the tyrosine kinase domain. By comparison, no EGFR mutations were observed in seven patients with non small cell lung cancer who had no response to gefitinib. To study the functional properties of the EGFR encoded by these mutated genes, EGFR with the L747–P753 in-frame deletion and EGFR with the L858R missense mutation were expressed in Cos-7 cells. In the absence of serum and of activating growth factors, neither wild-type nor mutant EGFR demonstrated autophosphorylation. However, EGF treatment caused a three- to four-fold increase in EGFR phosphorylation in both mutant EGFRs as compared with the activation of the wild-type EGFR. Moreover, the two mutant receptors had a continued activation for up to three hours. Remarkably, both mutant receptors were more sensitive than the wildtype receptor to inhibition by gefitinib. Since all the mutations are clustered near the ATP cleft of the tyrosine kinase domain, where they flank amino acids shown in crystallographic studies to mediate binding of 4-anilinoquinazoline compounds, such as gefitinib (Stamos et al. 2002), it is possible that the mutations result in repositioning of these critical residues, stabilizing their interaction with both ATP and its competitive inhibitor gefitinib. Such a mechanism could explain both the increased receptor activation after ligand binding and the enhanced inhibition induced by gefitinib. The effects of mutated variants of EGFR has also been investigated in other NSCLC cell lines, such as H1650, that carries the in-frame deletion E746-A750, or H1975, with missense mutation L858R, or NCI358, H1666 and H1734 that have wildtype EGFR (Sordella et al. 2004). Cell lines harbouring EGFR mutations displayed a selective activation of anti-apoptotic signals through Akt and signal transduction and activator of transcription (STAT) 5, which promote cell survival but have no effect on Erk, which induces proliferation. EGF-induced autophosphorylation of tyrosine992 (Y992) and tyrosine1068 (Y1068) was markedly elevated in the two cancer cell lines harboring EGFR gene mutations, with a concomitant increase in phosphorylation of Akt and STAT5 but not of Erk. The two lung cancer cell lines harboring EGFR mutations exhibited an increased proliferative activity as relative to lung cancer cells expressing wild-type EGFR when maintained in the presence of EGF in low serum concentrations. However, the proliferation rate and cell density at confluence were comparable at normal serum concentrations. Moreover, NSCLC cells expressing mutant EGFRs underwent extensive apoptosis after small interfering RNA (siRNA)–mediated knockdown of the mutant EGFR or treatment with pharmacological inhibitors of Akt and STAT signaling and were relatively resistant to apoptosis induced by conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. Thus, these mutant EGFRs selectively transduce survival signals on which NSCLCs become dependent and it is possible that NSCLCs expressing only wild-type receptors do not display a similar dependence on EGFR activation and this could account for the relative gefitinib-insensitivity of human NSCLC that express wild-type EGFR.
Somatic mutations in the EGFR TK domain have also been associated with sensitivity to erlotinib (Pao et al. 2004). Five of seven NSCLC tumor specimens from patients which were sensitive to erlotinib treatment had analogous somatic mutations (in-frame deletions within exon 19 or point mutations within exon 21), as opposed to no mutations found in 10 erlotinib-refractory tumors. Most EGFR mutation-positive tumors were adenocarcinomas from patients who have never smoked.
The issue of EGFR mutations in sensitivity/resistance to EGFR inhibitors has been addressed also for non-selective EGFR antagonists, such as ZD6474 (Arao et al. 2004). A strong correlation has been noted in several cancer cells lines in vitro between the IC50 values of gefitinib and ZD6474; conversely no correlation was observed between the sensitivity to ZD6474 and the level of EGFR or VEGFR expression. The NSCLC cell line PC-9 was hypersensitive to gefitinib and ZD6474, and a small (15-bp) in-frame deletion of the ATP-binding site (exon 19) in the EGFR TK domain was detected (delE746-A750–type deletion). The involvement of this EGFR mutation in the cellular sensitivity to ZD6474 has been confirmed by transfection of HEK293 cells with the EGFR mutated construct. These cells exhibited a 60-fold higher sensitivity to ZD6474 as compared with cells expressing wild-type EGFR. ZD6474 inhibited the phosphorylation of the mutant EGFR by 10-fold as compared with cells with wild-type EGFR. The correlation between ‘gain of function’, somatic EGFR mutations and sensitivity to small molecule EGFR-TKIs has been documented in NSCLC cell lines and patients. However, similar mutations have not been found or appear to be very rare in other cancer types, including breast, glioblastomas, CRC and HNSCC (Barber et al. 2004, Lynch et al. 2004, Lee et al. 2005).
Other mechanisms of cancer cell resistance to EGFR inhibitors
Cyclin D1 and p27KIP1 are downstream effectors involved in EGFR-dependent intracellular mitogenic signaling (Busse et al. 2001, Lenferink et al. 2001, Hulit et al. 2002), which are commonly deregulated in various cancers (Jares et al. 1994, Bova et al. 1999, Okami et al. 1999). The relationship between deregulated Cyclin D1 expression and sensitivity to gefitinib has been investigated to determine whether this frequently occurring oncogenic change could affect the cellular response to this EGFR-TKI (Kalish et al. 2004). In a panel of six EGFR-overexpressing HNSCC lines analyzed, three of them which were carrying Cyclin D1 gene amplification and/or protein overexpression displayed resistance to gefitinib. SCC9 HNSCC cells transfected with a Cyclin D1 expression vector had a sustained proliferation with a high Sphase fraction when treated with gefitinib, whereas empty vector control clones and the parental SCC 9 cells were significantly growth-inhibited with a marked reduction in S-phase. The resistance of Cyclin D1- overexpressing clones and Cyclin D1-amplified cell lines was associated with maintenance of Cyclin D1 protein expression after gefitinib treatment.
Recently, proteome pattern studies have been performed to understand the cellular signals underlying resistance to MAbs against EGFR. To define proteins involved in EGFR-triggered growth regulation and potential resistance mechanisms, the proteome profile of human colorectal cancer cell lines with high expression of functional EGFR but different sensitivity to cetuximab has been characterized (Skvortsov et al. 2004). Cetuximab treatment resulted in a complete saturation of EGFR in Caco-2 and HRT-18 CRC cell lines. However, whereas Caco-2 cells showed inhibition of proliferation, growth of HRT-18 cells was not suppressed. Using two-dimensional electrophoresis gels and subsequent mass spectrometry, the authors identified 14 proteins differentially expressed in the two cell lines. Among these proteins, fatty acid binding protein and heat shock protein 27 might contribute to the resistance of HRT-18 CRC cells to cetuximab treatment. These data demonstrate that proteome-based investigations could be an useful tool to better understand the complex protein interactions involved in EGFR signaling and in resistance to EGFR inhibitors.
Sensitivity to EGFR antagonists could be also related to genetic differences among individuals. The hypothesis that genetic variations in the EGFR gene could explain the resistant phenotype has been tested (Amador et al. 2004). In fact, several studies have revealed that polymorphic variations in genes encoding drug targets affect the response and toxicity to therapeutic agents (Arranz et al. 1995, Yoshida et al. 1995, Benetos et al. 1996, van Essen et al. 1996, Henrion et al. 1998, Lima et al. 1999). The EGFR gene contains a highly polymorphic sequence in intron 1, which consists of a variable number of CA dinucleotide repeats ranging from 9 to 21 (Chrysogelos 1993). This sequence has been shown to affect the efficiency of gene transcription such that subjects or cell lines with a greater number of CA repeats have lower levels of EGFR mRNA and protein expression (Gebhardt et al. 1999, Buerger et al. 2000). HNSCC lines with lower numbers of CA dinucleotides in the CA single sequence repeat (CA-SSR) of the intron 1 had a higher expression of EGFR and were more sensitive to the growth inhibitory effects of erlotinib. Phenotypic modification by silencing EGFR mRNA expression in HN029 cells sensitive to erlotinib induced resistance to this drug. The analysis of clinical specimens obtained from tumor and paired peripheral blood cells from 30 patients with advanced HNSCC revealed an equivalent number of CA dinucleotides between paired samples of the same individual, supporting the notion that this region is not commonly somatically mutated in the process of tumor development.
For some common anticancer agents, one possible mechanism of resistance is increased drug efflux resulting from the activity of membrane-associated pumps, such as P-glycoprotein (P-gp), the product of the multidrug resistance gene mdr-1. Since many small molecule inhibitors of tyrosine kinase have a neutral and hydrophobic nature, they could be substrates for P-gp or similar-acting efflux pumps. Intracellular accumulation of CI-1033, a small molecule EGFR-TKI, seems to be dependent on the breast cancer resistance protein (BCRP), a recently cloned ATP binding cassette transporter. In MDA-MB-231 breast cancer cells, transfection of BCRP resulted in a decrease in CI-1033 accumulation, compared with cells transfected with empty vector s(Erlichman et al. 2001). This observation suggests that CI-1033 is itself a substrate for BCRP, which in turn could possibly regulate the efflux of other specific EGFR inhibitors.
Conclusions
It is now well established that EGFR signaling is important to both normal development and to neoplastic transformation and that EGFR inhibition represents a valid anti-cancer approach. Different EGFR molecular antagonists have been evaluated in a clinical setting and some of these drugs have demonstrated dramatic clinical efficacy in a subset of NSCLC patients (in particular, gefitinib and erlotinib), as well as in HNSCC and colorectal cancer patients (in particular, cetuximab). However, constitutive and acquired resistance to EGFR inhibitors represent a major and unsolved clinical problem in treating cancer patients with EGFR expressing tumors. Since the enhanced and uncontrolled activation of key intracellular signal transduction pathways, the use of alternative growth factor receptor signals, the presence of EGFR gene alterations might constitute a common series of mechanisms which cause resistance to EGFR blockade, it is reasonable that only novel combined molecularly targeted therapeutic approaches aimed to block both the EGFR and these mechanisms may represent an effective anticancer treatment for a larger group of patients whose tumors have a functional EGFR pathway.
Acknowledgements
The research program in the laboratory of the authors was performed with a research grant from AIRC (Associazione Italiana per la Ricerca sul Cancro), Italy.
References
Ali IU, Schriml LM & Dean M 1999 Mutational spectra of PTEN/MMAC1 gene a tumor suppressor with lipid phosphatase activity. Journal of the National Cancer Institute 17 1922–1932.
Amador ML, Oppenheimer D, Perea S, Maitra A, Cusati G, Iacobuzio-Donahue C, Baker SD, Ashfaq R, Takimoto C, Forastiere A & Hidalgo M 2004 An epidermal growth factor receptor intron 1 polymorphism mediates response to epidermal growth factor receptor inhibitors. Cancer Research 15 9139–9143.
Arao T, Fukumoto H, Takeda M, Tamura T, Saijo N & Nishio K 2004 Small in-frame deletion in the epidermal growth factor receptor as a target for ZD6474. Cancer Research 15 9101–9104.
Arranz M, Collier D, Sodhi M, Ball D, Roberts G, Price J, Sham P & Kerwin R 1995 Association between clozapine response and allelic variation in 5-HT2A receptor gene. Lancet 346 281–282.
Arteaga CL 2002 Epidermal growth factor receptor dependence in human tumors more than just expression Oncologist 7 (Suppl 4) 31–39.
Arteaga CL & Baselga J 2003 Clinical trial design and end points for epidermal growth factor receptor-targeted therapies implications for drug development and practice. Clinical Cancer Research 9 1579–1589.
Azemar M, Schmidt M, Arlt F, Kennel P, Brandt B, Papadimitriou A, Groner B & Wels W 2000 Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapid tumor regression in vivo. International Journal of Cancer 15 269–275.
Barber TD, Vogelstein B, Kinzler KW & Velculescu VE 2004 Somatic mutations in colorectal cancer and glioblastomas. New England Journal of Medicine 351 2883.
Baselga J, Norton L, Albanell J, Kim YM & Mendelsohn J 1998 Recombinant humanized anti- HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Research 58 2825–2831.
Benetos A, Cambien F, Gautier S, Ricard S, Safar M, Laurent S, Lacolley P, Poirier O, Topouchian J & Asmar R 1996 Influence of the angiotensin II type 1 receptor gene polymorphism on the effects of perindopril and nitrendipine on arterial stiffness in hypertensive individuals. Hypertension 28 1081–1084.
Bianco R, Shin I, Ritter CA, Yakes FM, Basso A, Rosen N, Tsurutani J, Dennis PA, Mills GB & Arteaga CL 2003 Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors. Oncogene 8 2812–2822.
Bishop PC, Myers T, Robey R, Fry DW, Liu ET, Blagosklonny MV & Bates SE 2002 Differential sensitivity of cancer cells to inhibitors of the epidermal growth factor receptor family. Oncogene 3 119–127.
Blencke S, Ullrich A & Daub H 2003 Mutation of threonine 766 in the epidermal growth factor receptor reveals a hotspot for resistance formation against selective tyrosine kinase inhibitors. The Journal of Biological Chemistry 25 15435–15440.
Bova RJ, Quinn DI, Nankervis JS, Cole IE, Sheridan BF, Jensen MJ, Morgan GJ, Hughes CJ & Sutherland RL 1999 Cyclin D1 and p16INK4A expression predict reduced survival in carcinoma of the anterior tongue. Clinical Cancer Research 5 2810–2819.
Buerger H, Gebhardt F, Schmidt H, Beckmann A, Hutmacher K, Simon R, Lelle R, Boecker W & Brandt B 2000 Length and loss of heterozygosity of an intron 1 polymorphic sequence of egfr is related to cytogenetic alterations and epithelial growth factor receptor expression. Cancer Research 60 854–857.
Busse D, Doughty RS & Arteaga CL 2001 HER-2/neu (erbB- 2) and the cell cycle. Seminars in Oncology 27 (Suppl 11) 3–8.
Chakravarti A, Loeffler JS & Dyson NJ 2002 Insulin-like growth factor receptor I mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation of phosphoinositide 3-kinase signaling. Cancer Research 1 200–207.
Chan JM, Stampfer MJ, Giovannucci E, Gann PH, Ma J, Wilkinson P, Hennekens CH & Pollak M 1998 Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science 23 563–566.
Cheng JQ, Godwin AK, Bellacosa A, Taguchi T, Franke TF, Hamilton TC, Tsichlis PN & Testa JR 1992 AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. PNAS 1 9267–9271.
Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA, Watson DK & Testa JR 1996 Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. PNAS 16 3636–3641.
Chrysogelos SA 1993 Chromatin structure of the EGFR gene suggests a role for intron 1 sequences in its regulation in breast cancer cells. Nucleic Acids Research 21 5736–5741.
Ciardiello F, Damiano V, Bianco R, Bianco C, Fontanini G, De Laurentiis M, De Placido S, Mendelsohn J, Bianco AR & Tortora G 1996 Antitumor activity of combined blockade of epidermal growth factor receptor and protein kinase A. Journal of the National Cancer Institute 4 1770–1776.
Ciardiello F & Tortora G 2001 A novel approach in the treatment of cancer targeting the epidermal growth factor receptor. Clinical Cancer Research 7 2958–2970.
Ciardiello F, Caputo R, Troiani T, Borriello G, Kandimalla ER, Agrawal S, Mendelsohn J, Bianco AR & Tortora G 2001a Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis, and cooperate with cytotoxic drugs in human cancer cell lines. International Journal of Cancer 15 172–178.
Ciardiello F, Caputo R, Bianco R, Damiano V, Fontanini G, Cuccato S, De Placido S, Bianco AR & Tortora G 2001b Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clinical Cancer Research 7 1459–1465.
Ciardiello F, Bianco R, Caputo R, Caputo R, Damiano V, Troiani T, Melisi D, De Vita F, De Placido S, Bianco AR & Tortora G 2004 Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy. Clinical Cancer Research 15 784–793.
Clynes RA, Towers TL, Presta LG & Ravetch JV 2000 Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nature Medicine 6 443–446.
Erlichman C, Boerner SA, Hallgren CG, Spieker R, Wang XY, James CD, Scheffer GL, Maliepaard M, Ross DD, Bible KC & Kaufmann SH 2001 The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast cancer resistance protein-mediated drug efflux. Cancer Research 15 739–748.
Forgacs E, Biesterveld EJ, Sekido Y, Fong K, Muneer S, Wistuba II, Milchgrub S, Brezinschek R, Virmani A, Gazdar AF & Minna JD 1998 Mutation analysis of the PTEN/MMAC1 gene in lung cancer. Oncogene 24 1557–1565.
Gebhardt F, Zanker KS & Brandt B 1999 Modulation of epidermal growth factor receptor gene transcription by a polymorphic dinucleotide repeat in intron 1. The Journal of Biological Chemistry 274 13176–13180.
Gille J, Swerlick RA & Caughman SW 1997 Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation. EMBO J 17 750–759.
Goldman CK, Kim J, Wong WL, King V, Brock T & Gillespie GY 1993 Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells a model of glioblastoma multiforme pathophysiology. Molecular Biology of the Cell 4 121–133.
Grandis JR, Melhem MF, Gooding WE, Day R, Holst VA, Wagener MM, Drenning SD & Tweardy DJ 1998 Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival. Journal of the National Cancer Institute 90 824–832.
Henrion D, Amant C, Benessiano J, Philip I, Plantefeve G, Chatel D, Hwas U, Desmont JM, Durand G, Amouyel P & Levy BI 1998 Angiotensin II type 1 receptor gene polymorphism is associated with an increased vascular reactivity in the human mammary artery in vitro. Journal of Vascular Research 35 356–362.
Holly JM 1998 Insulin-like growth factor-I and new opportunities for cancer prevention. Lancet 351 1373–1375.
Huang SF, Liu HP, Li LH, Ku YC, Fu YN, Tsai HY, Chen YT, Lin YF, Chang WC, Kuo HP, Wu YC, Chen YR & Tsai SF 2004 High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clinical Cancer Research 15 8195–8203.
Hulit J, Lee RJ, Russell RG & Pestell RG 2002 ErbB-2-induced mammary tumor growth: the role of cyclin D1 and p27Kip1. Biochemical Pharmacology 64 827–836.
Jares P, Fernandez PL, Campo E, Nadal A, Bosch F, Aiza G, Nayach I, Traserra J & Cardesa A 1994 PRAD-1/cyclin D1 gene amplification correlates with messenger RNA overexpression and tumor progression in human laryngeal carcinomas. Cancer Research 1 4813–4817.
Jones HE, Goddard L, Gee JM, Hiscox S, Rubini M, Barrow D, Knowlden JM, Williams S, Wakeling AE & Nicholson RI 2004 Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells. Endocrine-Related Cancer 11 793–814.
Kalish LH, Kwong RA, Cole IE, Gallagher RM, Sutherland RL & Musgrove EA 2004 Deregulated cyclin D1 expression is associated with decreased efficacy of the selective epidermal growth factor receptor tyrosine kinase inhibitor gefitinib in head and neck squamous cell carcinoma cell lines. Clinical Cancer Research 15 7764–7774.
Kuan CT, Wikstrand CJ & Bigner DD 2001 EGF mutant receptor vIII as a molecular target in cancer therapy. Endocrine-Related Cancer 8 83–96.
Learn CA, Hartzell TL, Wikstrand CJ, Archer GE, Rich JN, Friedman AH, Friedman HS, Bigner DD & Sampson JH 2004 Resistance to tyrosine kinase inhibition by mutant epidermal growth factor receptor variant III contributes to the neoplastic phenotype of glioblastoma multiforme. Clinical Cancer Research 1 3216–3224.
Lee JW, Soung YH, Kim SY, Park WS, Nam SW, Lee JY, Yoo NJ & Lee SH 2005 Absence of EGFR mutations in the kinase domain in common human cancers beside non-small cell lung cancer. International Journal of Cancer 113 510–511.
Lenferink AE, Busse D, Flanagan WM, Yakes FM & Arteaga CL 2001 ErbB2/neu kinase modulates cellular p27(Kip1) and cyclin D1 through multiple signaling pathways. Cancer Research 1 6583–6591.
Lengauer C, Kinzler KW & Vogelstein B 1998 Genetic instabilities in human cancers. Nature 396 643–649.
LeRoith D, Werner H, Beitner-Johnson D & Roberts CT Jr 1995 Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocrine Reviews 16 143–163.
Li B, Chang CM, Yuan M, McKenna WG & Shu HK 2003 Resistance to small molecule inhibitors of epidermal growth factor receptor in malignant gliomas. Cancer Research 1 7443–7450.
Li DM & Sun H 1998 PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells. PNAS 22 15406–15411.
Lima JJ, Thomason DB, Mohamed MH, Eberle LV, Self TH & Johnson JA 1999 Impact of genetic polymorphisms of the beta2-adrenergic receptor on albuterol bronchodilator pharmacodynamics. Clinical Pharmacology and Therapeutics 65 519–525.
Lu Y, Lin YZ, LaPushin R, Cuevas B, Fang X, Yu SX, Davies MA, Khan H, Furui T, Mao M, Zinner R, Hung MC, Steck P, Siminovitch K & Mills GB 1999 The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene 25 7034–45.
Lu Y, Zi X & Pollak M 2004 Molecular mechanisms underlying IGF-I-induced attenuation of the growth-inhibitory activity of trastuzumab (Herceptin) on SKBR3 breast cancer cells. International Journal of Cancer 20 334–341.
Lu Y, Zi X, Zhao Y, Mascarenhas D & Pollak M 2001 Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). Journal of the National Cancer Institute 19 1852–1857.
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J & Haber DA 2004 Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. New England Journal of Medicine 20 2129–2139.
Ma YY, Wei SJ, Lin YC, Lung JC, Chang TC, Whang-Peng J, Liu JM, Yang DM, Yang WK & Shen CY 2000 PIK3CA as an oncogene in cervical cancer. Oncogene 25 2739–2544.
Mendelsohn J 2001 The epidermal growth factor receptor as a target for cancer therapy. Endorine-Related Cancer 8 3–9.
Moscatello DK, Holgado-Madruga M, Godwin AK, Ramirez G, Gunn G, Zoltick PW, Biegel JA, Hayes RL & Wong AJ 1995 Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Research 1 5536–5539.
Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, Klos KS, Li P, Monia BP, Nguyen NT, Hortobagyi GN, Hung MC & Yu D 2004 PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6 117–127.
Nicholson RI, Gee JM & Harper ME 2001 EGFR and cancer prognosis. European Journal of Cancer 37 (Suppl 4) 9–15.
Nickerson T, Chang F, Lorimer D, Smeekens SP, Sawyers CL & Pollak M 2001 In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR). Cancer Research 61 6276–6280.
Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK & Huang HJ 1994 A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. PNAS 2 7727–7731.
Nowell PC 1976 The clonal evolution of tumor cell populations. Science 194 23–28.
Okami K, Reed AL, Cairns P, Koch WM, Westra WH, Wehage S, Jen J & Sidransky D 1999 Cyclin D1 amplification is independent of p16 inactivation in head and neck squamous cell carcinoma. Oncogene 10 3541–3545.
Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE & Meyerson M 2004 EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 4 1497–500.
Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, Singh B, Heelan R, Rusch V, Fulton L, Mardis E, Kupfer D, Wilson R, Kris M & Varmus H 2004 EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. PNAS 7 13306–13311.
Petit AM, Rak J, Hung MC, Rockwell P, Goldstein N, Fendly B & Kerbel RS 1997 Neutralizing antibodies against EGF and ErbB-2/neu receptor tyrosine kinases down-regulate VEGF production by tumor cells in vitro and in vivo Angiogenic implications for signal transduction therapy of solid tumors. American Journal of Pathology 151 1523–1530.
Pietras RJ, Pegram MD, Finn RS, Maneval DA & Slamon DJ 1998 Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs. Oncogene 17 2235–2249.
Resnicoff M & Baserga R 1998 The role of the insulin-like growth factor I receptor in transformation and apoptosis. Annals of the New York Academy of Sciences 842 76–81.
Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, Lai JL, Philippe N, Facon T, Fenaux P & Preudhomme C 2002 Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 100 1014–1018.
Roumiantsev S, Shah NP, Gorre ME, Nicoll J, Brasher BB, Sawyers CL & Van Etten RA 2002 Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. PNAS 6 10700–10705.
Rusch V, Baselga J, Cordon-Cardo C, Orazem J, Zaman M, Hoda S, McIntosh J, Kurie J & Dmitrovsky E 1993 Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Research 15 (Suppl 10) 2379–2385.
Salomon DS, Brandt R, Ciardiello F & Normanno N 1995 Epidermal growth factor-related peptides and their receptors in human malignancies. Critical Reviews in Oncology/Hematology 19 183–232.
Shayesteh L, Lu Y, Kuo WL, Baldocchi R, Godfrey T, Collins C, Pinkel D, Powell B, Mills GB & Gray JW 1999 PIK3CA is implicated as an oncogene in ovarian cancer. Nature Genetics 21 99–102.
She QB, Solit D, Basso A & Moasser MM 2003 Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'-kinase/Akt pathway signaling. Clinical Cancer Research 1 4340–4346.
Skvortsov S, Sarg B, Loeffler-Ragg J, Skvortsova I, Lindner H, Werner Ott H, Lukas P, Illmensee K & Zwierzina H 2004 Different proteome pattern of epidermal growth factor receptor-positive colorectal cancer cell lines that are responsive and nonresponsive to C225 antibody treatment. Molecular Cancer Therapeutics 3 1551–1558.
Sordella R, Bell DW, Haber DA & Settleman J 2004 Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 20 1163–1167.
Stamos J, Sliwkowski MX & Eigenbrot C 2002 Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. The Journal of Biological Chemistry 29 46265–46272.
Taguchi F, Koh Y, Koizumi F, Tamura T, Saijo N & Nishio K 2004 Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib (‘Iressa’)-sensitive and resistant xenograft models. Cancer Science 1 984–989.
van Essen GG, Rensma PL, de Zeeuw D, Sluiter WJ, Scheffer H, Apperloo AJ & de Jong PE 1996 Association between angiotensinconverting-enzyme gene polymorphism and failure of renoprotective therapy. Lancet 347 94–95.
Viloria-Petit A, Crombet T, Jothy S, Hicklin D, Bohlen P, Schlaeppi JM, Rak J & Kerbel RS 2001 Acquired resistance to the antitumor effect of epidermal growth factor receptorblocking antibodies in vivo A role for altered tumor angiogenesis. Cancer Research 61 5090–5101.
Viloria-Petit AM & Kerbel RS 2004 Acquired resistance to EGFR inhibitors mechanisms and prevention strategies. International Journal of Radiation Oncology Biology Physics 1 914–926.
Vivanco I & Sawyers CL 2002 The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nature Reviews Cancer 2 489–501.
Wells A 1999 EGF receptor. The International Journal of Biochemistry and Cell Biology 31 637–643.
Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S & Arteaga CL 2002 Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Research 15 4132–4141.
Yamazaki H, Kijima H, Ohnishi Y, Abe Y, Oshika Y, Tsuchida T, Tokunaga T, Tsugu A, Ueyama Y, Tamaoki N & Nakamura M 1998 Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression. Journal of the National Cancer Institute 15 581–587.
Yoshida H, Mitarai T, Kawamura T, Kitajima T, Miyazaki Y, Nagasawa R, Kawaguchi Y, Kubo H, Ichikawa I & Sakai O 1995 Role of the deletion of polymorphism of the angiotensin converting enzyme gene in the progression and therapeutic responsiveness of IgA nephropathy. The Journal of Clinical Investigation 96 2162–2169.
Yu H & Rohan T 2000 Role of the insulin-like growth factor family in cancer development and progression. Journal of the National Cancer Institute 92 1472–1489.(Roberto Bianco, Teresa Tr)