International Journal of Radiation Oncology*Biology*Physics
EGFR INHIBITORSThe contribution of epidermal growth factor receptor (EGFR) signaling pathway to radioresistance in human gliomas: a review of preclinical and correlative clinical data
Introduction
During the past decade, the receptor tyrosine kinase (RTK) family of growth factor receptors has been found to play a major role in the pathogenesis of many human malignancies, including malignant gliomas. The epidermal growth factor receptor (EGFR) is a 170-kd RTK that is composed of an extracellular binding domain, a transmembrane lipophilic segment, and an intracellular domain that has protein kinase activity 1, 2, 3. The ligands for EGFR include the epidermal growth factor as well as transforming growth factor α. After binding of ligand, EGFR dimerizes, which activates the intrinsic protein tyrosine kinase and triggers a cascade of downstream signaling events, as described below.
Of the 4 EGFR family members, erbB1-B4, erbB1 has been the best characterized to date. It has been observed that EGFR gene amplification (located on chromosome 7) is quite common in glioblastomas (GBMs), occurring in up to 50% of cases (4). In lower-grade gliomas, EGFR gene amplification has been found to be far less common. Common molecular anomalies associated with various stages of gliomagenesis are illustrated in Fig. 1. Approximately 40% of the GBMs with EGFR amplification express a mutant form of EGFR, referred to as EGFRvIII. The EGFRvIII mutant lacks a portion of the extracellular ligand-binding domain as the result of genomic deletions of exons 2–7 in the EGFR mRNA 5, 6. This results in constitutive phosphorylation, or activation, of the EGFR pathway. Introduction of EGFRvIII into glioma cells has been shown to enhance cell proliferation and invasion, while inhibiting apoptosis 7, 8, 9. Additionally, it has been demonstrated to confer resistance to chemotherapeutic agents via upregulation of Bcl-XL expression (10).
There is increasing evidence that EGFR plays an important role in normal astrocyte development and differentiation. Indeed, understanding fundamental mechanisms by which EGFR enhances survival in progenitor cells has the potential to shed light on how EGFR enhances survival in glioma cells in response to radiation. It has been reported that highest levels of EGFR expression occur with the peak of gliogenesis in the embryonic and early perinatal period, suggesting an association with astrocyte and/or oligodendrocyte development 11, 12. There are several lines of evidence directly associating EGFR with the glial differentiation process. First, targeted deletion of EGFR in mice results in embryonic or perinatal lethality with the affected mice demonstrating cortical dysgenesis and reduced numbers of astrocytes 13, 14, 15. Second, retroviral-mediated overexpression of EGFR in the early ventricular zone results in proliferation of stem cells as well as premature astrocyte differentiation (11). Third, although cultured neural stem cells preferentially differentiate into astrocytes upon transplantation into the adult brain, the transplanted neural stem cells remain undifferentiated and continue to proliferate if epidermal growth factor is simultaneously infused into the lateral ventricles (16).
These results, taken together, suggest that EGFR seems to have a role in normal astrocyte differentiation and survival of the neural stem cell compartment. It is entirely possible that increased EGFR pathway activation may interfere with the normal differentiation process and serve to enhance malignant potential in gliomas. Because the net effect of EGFR pathway activation, as illustrated by these models, is to enhance cellular survival, it is not surprising that there is increasing evidence that EGFR signaling is associated with resistance to conventional cytotoxic agents, as described below.
There is strong evidence suggesting that EGFR plays a key role in contributing to radiation resistance of malignant gliomas. These results have emerged from EGFR inactivation studies using one of several approaches: monoclonal antibodies, tyrosine kinase inhibitors, antisense oligonucleotides, dominant-negative EGFR mutants, etc. These reports suggest that when EGFR antagonism is combined with radiation, a significant radiosensitization effect is observed 17, 18, 19, 20, 21, 22. In one study, glioma cells were treated with an adenoviral construct containing dominant-negative EGFR (Ad-EGFR-CD533) 19, 21. A dose enhancement ratio of 1.85 was observed. The authors suggest that because Ad-EGFR-CD533 inhibits the activation of all ErbB receptors by preventing functional receptor heterodimerization and transphosphorylation, there may be at least a theoretical advantage in combining Ad-EGFR-CD533 with other types of EGFR inhibitors. However, another report suggested that not all EGFR-expressing GBMs are amenable to anti-EGFR radiosensitization and that EGFR expression levels may not predict which tumors respond best to this treatment strategy (23). In this study, 2 primary GBM cell lines with equivalent EGFR expression levels were found to have very different sensitivities to the EGFR tyrosine kinase inhibitor, AG1478. This was apparent despite similar reductions in EGFR signaling in both cell lines, as measured by phospho-EGFR levels. It was found that the resistant GBM cell line demonstrated an upregulation of insulin-like growth factor receptor 1 (IGFR1) levels upon AG1478 administration. This resulted in sustained signaling through the phosphatidylinositol 3-kinase (PI3-K)/AKT pathway and ultimately in resistance to AG1478. Cotargeting IGFR1 with EGFR greatly enhanced both spontaneous and radiation-induced apoptosis and the invasive potential of this resistant cell line. AKT and p70s6k seemed to be important downstream targets of IGFR1-mediated resistance to anti-EGFR targeting. These findings suggest that IGFR1 signaling through PI3-K may represent a novel and potentially important mechanism of resistance to anti-EGFR therapy.
EGFR has distinct downstream signaling pathways that have been previously reported to play important roles in radiation resistance. These downstream pathways are illustrated in Fig. 2. The RAS/RAF/MAPK cascade is one of the best characterized EGFR-regulated pathways. RAS is a guanine nucleotide-binding protein, which in its GTP-bound form results in the downstream activation of numerous important signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway. RAS-GTP is inactivated to RAS-GDP through intrinsic GTPase activity, catalyzed by GTPase-activating proteins. Oncogenic RAS is resistant to RAS GTPase-activating proteins and is therefore locked into its GTP-bound form, resulting in constitutive activation. Although oncogenic RAS has been found to be common in other tumor types, it has not typically been observed in human gliomas 24, 25. However, it has been reported that high-grade gliomas commonly have elevated levels of RAS-GTP 26, 27, 28, 29, 30, 31, 32. Because gliomas commonly have amplification/overexpression of RTKs such as EGFR, PDGFR, IGFR1, etc., this may represent an important mechanism through which RAS signaling is upregulated in gliomas. It has been demonstrated that antagonizing RAS either by using dominant-negative constructs or farnesyltransferase inhibitors results in antiproliferative, antiangiogenic, and proapoptotic effects in glioma cell lines in vitro27, 28, 29, 30, 31. Combining farnesyltransferase inhibitors with chemotherapy was found to result in ∼69% growth inhibition in preclinical in vivo models (31). Furthermore, it has been demonstrated that astrocyte-specific expression of activated RAS results in malignant astrocytoma formation in a transgenic mouse model of human gliomas. Therefore, given that RAS is a major effector of EGFR signaling, this may represent one of the major mechanisms by which EGFR enhances the malignant potential of glioma cells.
To further support this observation, a recent study reported that EGFR may play a critical role also in mediating resistance to sequential administration of radiation and chemotherapy in primary human glioblastoma cells in a RAS-dependent manner (18). It is well known (and a point of frustration) that, unlike treatment of other types of tumors, treatment of glioblastoma with combined modality therapy involving chemotherapy and radiation has failed to appreciably improve outcome for patients compared to radiation alone. In 3 primary GBM cell lines, an actual antagonistic effect was observed between radiation and BCNU chemotherapy. These cell lines were coincidentally found to have strong expression of EGFR. Upon inhibition of EGFR with AG1478 (an EGFR tyrosine kinase inhibitor), it was found that this cross-resistance between sequentially administered radiation and BCNU was effectively abrogated. It was found that BCNU inhibited radiation-induced apoptosis through EGFR-mediated activation of PI3-K/AKT via RAS. On the other hand, radiation was found to inhibit BCNU-induced apoptosis through EGFR-mediated activation of both PI3-K and MAPK (p44/p42) pathways, also via RAS. Inhibition of either EGFR or RAS activity seems not only to abrogate the observed antagonism between sequentially administered radiation and chemotherapy, but also actually results in a greater enhancement of apoptosis in the setting of combined modality therapy than when administered with either radiation or chemotherapy as single agents. Therefore, these findings suggest that strategies to inactivate EGFR or RAS signaling may be critical to improving the efficacy of not only single-agent therapy, but also of combined modality therapy in gliomas.
There is increasing evidence that the observed role of EGFR in radiation resistance in preclinical models translates into adverse clinical outcome. In a study from the University of California at San Francisco, it was reported that positive EGFR immunoreactivity was associated with poor radiographically assessed radiation response (p = 0.046) (33). Thirty-three percent of tumors with no EGFR immunoreactivity had good radiation responses (>50% reduction in tumor size by CT or MRI), compared to 18% of tumors with intermediate EGFR staining and 9% of tumors with strong staining. In other studies, it has been demonstrated that EGFR immunostaining is of independent prognostic value in gliomas.
In another study, multivariate analysis was performed on clinical and biologic prognostic factors predictive of glial tumor outcome. A multivariate analysis including age, histology, tumor resection, EGFR immunostaining, and labeling index revealed that EGFR, labeling index, and tumor resection were the only independent significant predictors of survival (34). In another study, multivariate analysis of EGFR immunostaining was performed with other variables, including immunostaining for proliferating cell nuclear antigen, p53, bcl-2, and for apoptotic index (35). It was found that only EGFR positivity and apoptotic index were significant on multivariate analysis (p = 0.0053 and p = 0.0039, respectively). Quantitative measurements of EGFR expression in gliomas also seem to support previously reported immunohistochemical observations. EGFR levels in frozen glioma specimens were determined quantitatively through Western blotting and image analysis and were found to be significantly correlated with reduced survival (p < 0.0001) (36).
Studies on EGFR gene amplification also seem to suggest an association with both adverse radiation response, as determined radiographically, and association with more malignant histologies 37, 38. In a study from the University of California at San Francisco, comparative genomic hybridization was performed on 30 frozen GBM specimens (37). These cases were designated as either radioresistant or radiosensitive based on imaging criteria. Radiosensitive tumors were those that demonstrated greater than 50% reduction in contrast-enhanced volume after radiation; radioresistant tumors were those that demonstrated greater than 50% increase in contrast-enhanced volume postradiation. It was observed that a gain of chromosomes 7 (locus of EGFR gene) and 19 simultaneously was found in 30% of radioresistant cases, but gains of these chromosomes were absent from radiosensitive cases (p = 0.05). These data provide compelling evidence that upregulation of EGFR is directly associated with radioresistance in vivo as well as with the reduced patient survivals reported in prior studies.
Section snippets
Conclusions and future directions
EGFR seems to play a critical role in enhancing the malignant behavior of gliomas. Preclinical data suggest that EGFR regulates a number of important signaling pathways, including the RAS-RAF-MAPK cascade as well as the PI3-K/AKT pathway, which, when upregulated via EGFR amplification/overexpression, lead to increased cellular proliferation, migration, and invasion. It is also becoming clear that upregulation of EGFR may contribute to the intrinsic radioresistance of glioma cells. It is
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