Molecular radiobiology
Dual targeting of EGFR and focal adhesion kinase in 3D grown HNSCC cell cultures

https://doi.org/10.1016/j.radonc.2011.06.006Get rights and content

Abstract

Purpose

Epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) show frequent overexpression and hyperactivity in various human malignancies including head and neck squamous cell carcinomas (HNSCC). To examine effects of dual EGFR/FAK inhibition on cellular radiosensitivity of HNSCC cells in a more physiological environment, we employed a previously established laminin-rich extracellular matrix (lrECM) based three-dimensional (3D) cell culture model.

Materials and methods

UTSCC15 and SAS HNSCC cell lines stably transfected with EGFR-CFP or CFP were used. Single or combined EGFR (Cetuximab, siRNA) and FAK (TAE226, siRNA) inhibition were accomplished prior to measuring clonogenic survival and protein expression and phosphorylation. Immunofluorescence enabled visualization of EGFR-CFP and FAK.

Results

Cetuximab resulted in higher radiosensitization in EGFR-CFP overexpressing cell lines than CFP controls. Single EGFR or FAK inhibition mediated radiosensitization, while dual EGFR/FAK targeting further augmented this effect. Despite signaling alterations upon Cetuximab and siRNA knockdown, analysis of protein expression and phosphorylation indicates EGFR and FAK signaling coexistence without obvious overlap.

Conclusions

Combined EGFR/FAK targeting yielded stronger radiosensitization than either approach alone, which might be based on non-overlapping downstream signaling. Whether dual targeting of EGFR and FAK can reasonably be combined with radiotherapy and chemotherapy needs clarification.

Section snippets

Antibodies and reagents

Antibodies against EGFR Y1068, JNK T183/Y185, AlexaFluor 594 anti-mouse (Invitrogen), EGFR, Akt, Akt S473, Akt T308, p130Cas Y165, Src Tyr416, Src, ERK1/2, ERK1/2 Thr202/Tyr204, FAK Y397, FAK (for WB), JNK, Paxillin Y118, Paxillin (Cell Signaling), FAK (for IF #05-537), p130Cas (Millipore), β-actin (Sigma) and horseradish peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse (Amersham) antibodies were purchased as indicated. Matrigel (laminin-rich extracellular matrix, lrECM) was from

3D colony formation assay

Clonogenic survival under 3D conditions was measured as published [21], [32], [40]. In brief, DMEM with 10% FCS and 1%NEAA was mixed with lrECM to a final concentration of 0.5 μg/μl lrECM and used for the seeding of single cells in 1% agarose-coated 96-well plates. After 24 h, cells were treated with Cetuximab (0–5 μg/ml) and/or TAE226 (Novartis; 0.3 μM; control: DMSO) for 24 h and irradiated (X-ray, 0–6 Gy). After 26 h treatment with TAE226 or DMSO (2 h after irradiation), cells were washed six times

3D immunofluorescence staining

3D cultured cells were treated with AlexaFluor 568-labeled Cetuximab (0–5 μg/ml) for 1 h and then fixed with 3% formaldehyde/PBS in lrECM for 15 min. After transfer to a centrifugation tube, cells were permeabilized with 0.25% Triton X-100/PBS for 10 min, washed with PBS and blocked with 1% BSA/PBS for 30 min. Staining of FAK was performed with specific anti-FAK antibody for 2 h. After washing with PBS, AlexaFluor 594 anti-mouse antibody was added for 1 h. Cells were washed and dropped on a

Stable overexpression of EGFR-CFP failed to alter phosphorylation of putative downstream targets in 3D HNSCC cell cultures

At first, we evaluated the expression and phosphorylation of EGFR and downstream targets MAPK and Akt1 in stably EGFR-CFP transfected UTSCC15 and SAS cells grown in 3D lrECM. Under exponential and 10% FCS growth conditions, endogenous EGFR and exogenous EGFR-CFP were phosphorylated cell line-dependent (Fig. 1A). No significant modulation in the phosphorylation of the putative downstream targets Akt1 and ERK1/2 could be detected in EGFR-CFP overexpressing cells (Fig. 1A). Inhibitory efficacy and

Discussion

EGFR and FAK show frequent overexpression and hyperactivity in a variety of human malignancies including HNSCC. Unmet expectations within the in vitro-clinical translation of EGFR inhibitory approaches require additional investigations in tumor cell culture models better reflecting physiological growth conditions. In ECM-based 3D cell culture systems, as used here, integrin-mediated cell-ECM interactions and their associated signaling pathways become of exceptional importance [45], [47], [48].

Acknowledgments

This work was supported in part by the Bundesministerium für Bildung und Forschung (BMBF-03ZIK041 to N.C.) and the Medical Faculty Carl Gustav Carus, Dresden University of Technology, Germany (MeDDrive program to I.E.). The authors thank Dr. L. E. Samelson (National Cancer Institute, Bethesda, USA) for the pEGFR-CFP plasmid, Dr. R. Grenman (Turku University Central Hospital, Finland) for UTSCC15 and SAS cell lines, Novartis (Switzerland) for the FAK inhibitor TAE226, and Ms. C. Förster for

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