Molecular radiobiologyDual targeting of EGFR and focal adhesion kinase in 3D grown HNSCC cell cultures
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
References (50)
Chemoradiotherapy of head and neck cancer–can the bumble bee fly?
Radiother Oncol
(2009)- et al.
Epidermal growth factor receptor targeting in cancer
Semin Oncol
(2006) - et al.
ErbB receptors and signaling pathways in cancer
Curr Opin Cell Biol
(2009) - et al.
EGFR-targeted anti-cancer drugs in radiotherapy: preclinical evaluation of mechanisms
Radiother Oncol
(2007) - et al.
ErbB2 expression through heterodimerization with erbB1 is necessary for ionizing radiation- but not EGF-induced activation of Akt survival pathway
Radiother Oncol
(2010) - et al.
EGFR-TK inhibition before radiotherapy reduces tumour volume but does not improve local control: differential response of cancer stem cells and nontumourigenic cells?
Radiother Oncol
(2007) - et al.
Concurrent cetuximab, cisplatin, and radiation for squamous cell carcinoma of the head and neck in vitro
Radiother Oncol
(2009) - et al.
Reverse resistance to radiation in KYSE-150R esophageal carcinoma cell after epidermal growth factor receptor signal pathway inhibition by cetuximab
Radiother Oncol
(2009) - et al.
Radiotherapy plus cetuximab for locoregionally advanced head and neck cancer: 5-year survival data from a phase 3 randomised trial, and relation between cetuximab-induced rash and survival
Lancet Oncol
(2010) - et al.
Locoregionally advanced head and neck cancer treated with primary radiotherapy: a comparison of the addition of cetuximab or chemotherapy and the impact of protocol treatment
Int J Radiat Oncol Biol Phys
(2008)
A PAK-activated linker for EGFR and FAK
Dev Cell
Antiproliferative effects of EGFR tyrosine kinase inhibition and radiation-induced genotoxic injury are attenuated by adhesion to fibronectin
Radiother Oncol
Focal adhesion kinase (FAK) gene amplification and its clinical implications in gastric cancer
Hum Pathol
SRC-3Delta4 mediates the interaction of EGFR with FAK to promote cell migration
Mol Cell
3D cell cultures of human head and neck squamous cell carcinoma cells are radiosensitized by the focal adhesion kinase inhibitor TAE226
Radiother Oncol
Beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium
Cancer Cell
Radiation-induced EGFR-signaling and control of DNA-damage repair
Int J Radiat Biol
Molecular determinants of anti-EGFR sensitivity and resistance in metastatic colorectal cancer
Br J Cancer
Effect of cetuximab and fractionated irradiation on tumour micro-environment
Radiother Oncol
An essential role of integrin-linked kinase in the cellular radiosensitivity of normal fibroblasts during the process of cell adhesion and spreading
Int J Radiat Biol
Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase
J Cell Biol
Pharmacological inhibition of EGFR tyrosine kinase affects ILK-mediated cellular radiosensitization in vitro
Int J Radiat Biol
Cell adhesion-mediated radioresistance (CAM-RR) Extracellular matrix-dependent improvement of cell survival in human tumor and normal cells in vitro
Strahlenther Onkol
PINCH1 regulates Akt1 activation and enhances radioresistance by inhibiting PP1alpha
J Clin Invest
Cited by (40)
Adaptive Responses to Monotherapy in Head and Neck Cancer: Interventions for Rationale-Based Therapeutic Combinations
2019, Trends in CancerCitation Excerpt :Dephosphorylation of FAK, leading to the dissociation of the FAK/cortactin protein complex, enhances radiosensitization in HNC in vitro and in vivo [29]. Knockdown or inhibition of FAK or dual targeting of the EGFR and FAK enhance radiosensitivity in HNC [30,31]. Skvortsov et al. reported that the enhanced expression, activity, and nuclear localization of the Rac1 protein in tumor cells is a risk factor for tumor recurrence in HNC patients after chemoradiotherapy [32], and radioresistant HNC cell lines displayed Rac1 upregulation [33].
3D in vitro models of tumors expressing EGFR family receptors: a potent tool for studying receptor biology and targeted drug development
2019, Drug Discovery TodayCitation Excerpt :So, inhibition of MET and RON tyrosine kinases with crizotinib restored the sensitivity of the cells to cetuximab. In several studies, 3D tumor models in matrices were used to study the radiosensitivity of tumors [100–103]. In particular, it was demonstrated that the combined blocking of membrane proteins EGFR and FAK increased the radiosensitivity of HNSCC cell lines UTSCC15 and SAS more than separate inhibition of these proteins did [100].
EGFR and β1-integrin targeting differentially affect colorectal carcinoma cell radiosensitivity and invasion
2015, Radiotherapy and OncologyFocal adhesion signaling and therapy resistance in cancer
2015, Seminars in Cancer BiologyAcquired resistance to cetuximab is associated with the overexpression of Ras family members and the loss of radiosensitization in head and neck cancer cells
2013, Radiotherapy and OncologyCitation Excerpt :In the current study, we showed that cetuximab radiosensitizes UT5 cells but not SAS cells. A similar result has been reported previously for SAS cells in vitro [45]. In contrast to these in vitro data, Gurtner et al. [8] have shown that cetuximab in combination with fractionated radiotherapy improves local tumor control in SAS xenografts in vivo but not in UT5 xenografts in vivo.