Elsevier

Radiotherapy and Oncology

Volume 72, Issue 3, September 2004, Pages 267-273
Radiotherapy and Oncology

Radiation-induced activation of a common variant of EGFR confers enhanced radioresistance

https://doi.org/10.1016/j.radonc.2004.07.004Get rights and content

Abstract

Background and purpose

The type-III EGFR variant (EGFRvIII) is known to promote enhanced tumorigenicity. We have previously defined the importance of EGFRvIII in cellular radiation responses using Chinese hamster ovary cells (CHO). In the current study, we have extended our investigations of EGFRvIII to human tumor cells in vitro and in vivo and further verified the important role of EGFRvIII in modulating radiosensitivity.

Material and methods

The cell lines MDA-MB-231, U-87 MG, A-431 and U-373 MG were used. Adenoviral (Ad) vectors were produced to overexpress EGFRvIII in vitro or in xenograft tumors in vivo. The EGFR, EGFRvIII expression and tyrosine phosphorylation (Tyr-P) levels were quantified by Western blotting. The relative radiosensitivities were assessed in vitro by standard colony formation and in vivo by tumor growth delay assays.

Results

The presence of EGFRvIII was verified in all xenograft tumors tested with no detectable expression in the corresponding cells under in vitro culture conditions. MDA-MB-231 xenograft tumors demonstrated EGFRvIII expression levels, which were 1.9-fold higher relative to EGFRwt compared to a 14.5-fold higher Tyr-P. Ionizing radiation of these tumors at 4 Gy induced an average 3.2-fold increase in EGFRvIII Tyr-P. EGFRvIII expression in U-373 MG cells significantly enhanced survival after 4 Gy, which was completely abolished by dominant-negative EGFR-CD533. Finally, the ability of EGFRvIII to accelerate tumor growth during irradiation was confirmed in vivo.

Conclusion

EGFRvIII is frequently expressed in a variety of different tumor types and can confer significant radioresistance, thus further providing evidence for EGFRvIII as an additional important target in our approaches to radiosensitize malignant solid tumors.

Introduction

As part of neoplastic progression, increased expression of EGFR is often associated with the expression of variant species [8], [19]. Of these variants, the type-III EGFR (EGFRvIII) is most commonly expressed in human tumors [8]. EGFRvIII lacks a major portion of the Cys-rich ligand-binding domain near the NH2-terminus of the extracellular portion of the molecule [20]. EGFRvIII functions as an oncoprotein [9], [19], [28], [29] that promotes soft agar focus formation and tumorigenesis in nude mice [2]. Despite the knowledge of its transforming activity and its effects on signal transduction pathways in tumor cells [1], [8], [15], [18], the phenotypical and functional consequences of EGFRvIII expression on cellular radiation responses haven't been investigated in detail.

We have previously demonstrated in mechanistical studies using CHO cells that the presence of EGFRvIII confers a stronger radiation response than EGFR wild-type (wt) via the cytoprotective MAPK and PI3K pathways. Therefore, we now investigate in a more clinically related setting by using human tumor cell lines overexpressing EGFRvIII through adenoviral transduction, whether the presence of EGFRvIII also modulates radiosensitivity in tumor cells in vitro and in vivo including its inhibition through dominant-negative EGFR-CD533. Furthermore, in the current study we also applied our investigations to the endogenous EGFRvIII in established xenograft tumors and its induction through ionizing radiation.

Section snippets

Reagents

Unless otherwise listed, all reagents were obtained from Sigma Chemical Co. (St. Louis, MO). All electrophoresis reagents were from BioRad (Hercules, CA). Media and antibiotics were from GIBCO-BRL (Rockville, MD), and fetal bovine serum (FBS) was from Intergen (Purchase, NY). The following monoclonal antibodies (mAbs) were used: the anti-EGFR mAb cocktail (Ab14; Neo Markers; Fremont, CA), the anti-EGFRvIII mAb, DH8.3 (AbCam Limited, Cambridge, UK), the anti-EGFR mAb, Ab5, the anti-phospho-Tyr

EGFRvIII expression in xenograft tumors

EGFRvIII is expressed in a large variety of different human tumors [17]. In order to study the functional consequences of EGFRvIII expression on radiation responses, we first defined its expression in a variety of different tumor cell lines. For this we verified, by Western blot analyses, the presence of EGFRvIII protein in xenograft tumors relative to cells maintained in vitro. EGFRvIII expression was quantified in extracts from freshly excised tumors, thus reflecting the expression levels in

Discussion

The data presented demonstrate that EGFRvIII is widely expressed in xenograft tumors derived from a variety of different tumor cell types. This expression of EGFRvIII in vivo is restricted to cells growing in vivo as tumors. The high levels of EGFRvIII Tyr-P, relative to those of EGFR wt, reflect the constitutive activity. However, radiation exposure resulted in a pronounced stimulation of EGFRvIII in vitro and in vivo, leading to enhanced clonogenic survival in vitro and rapid tumor growth in

Acknowledgements

This work was supported by Public Health Service grants P01CA72955 and R01CA65896 (R.S.-U.) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, by the Florence and Hyman Meyers Head and Neck Cancer Research Fund and the Deutsche Krebshilfe, Dr Mildred Scheel Stiftung (G.L.).

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