Elsevier

Radiotherapy and Oncology

Volume 108, Issue 3, September 2013, Pages 473-478
Radiotherapy and Oncology

Epidermal growth factor receptor
Acquired resistance to cetuximab is associated with the overexpression of Ras family members and the loss of radiosensitization in head and neck cancer cells

https://doi.org/10.1016/j.radonc.2013.06.023Get rights and content

Abstract

Purpose

Cetuximab in combination with radiation therapy is used to treat patients with head and neck squamous cell carcinoma (HNSCC). In the present study, the mechanism of acquired resistance to cetuximab in HNSCC cells was investigated in vitro.

Material and methods

The HNSCC cell lines UT5 and SAS and UT5 cells with acquired resistance to cetuximab (UT5R9) were used. The radiotoxicity potentials of cetuximab and inhibitors of PI3K, MAPK and farnesylation were tested using a clonogenic survival assay. Western blotting was used to evaluate protein expression. The levels of EGFR ligands were detected by ELISA.

Results

Cetuximab inhibited proliferation and induced radiosensitization in UT5 cells but not in SAS cells. In comparison with UT5 cells, cetuximab-resistant SAS cells markedly overexpressed the K-Ras, H-Ras and N-Ras proteins, as detected by Western blotting. Resistance in UT5R9 cells was associated with the overexpression of the K-Ras, H-Ras and N-Ras proteins as well as an increase in the autocrine production of the EGFR ligands amphiregulin and transforming growth factor α (TGFα). UT5R9 cells were significantly more radioresistant than UT5 cells. Radioresistant UT5R9 cells were not radiosensitized by cetuximab, but knocking down H-RAS and N-RAS with siRNA and targeting Ras farnesylation using the farnesyltransferase inhibitor lonafarnib induced radiosensitization in these cells. Targeting PI3K and MEK revealed that the activation of the PI3K/Akt pathway but not the MAPK/ERK pathway is associated with radioresistance in UT5R9 cells.

Conclusion

Targeting Ras and PI3K activity improves the outcome of irradiation in cetuximab-resistant HNSCC cell lines in vitro.

Section snippets

Reagents

The antibodies against phospho-Akt (Ser-473), Akt, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, EGFR, actin, K-Ras and GAPDH; the control-siRNA and K-RAS-siRNA; the PI3K inhibitor LY294002; the MEK inhibitor PD98059; and the ELISA kits for TGFα and AREG have been previously described [32], [33], [34], [35], [36].

The EGFR tyrosine kinase inhibitor gefitinib was provided by Selleck Chemicals (Houston, TX, USA). Erlotinib was provided from Hoffmann-La Roche Ltd. (Basel, Switzerland), and cetuximab was

Acquired resistance to cetuximab is associated with increased proliferation and post-irradiation survival as well as the loss of cetuximab-induced radiosensitization

The effects of cetuximab on the proliferation and clonogenic activity of the HNSCC cell lines UT5 and UT5R9 (UT5 cells with acquired resistance to cetuximab after treatment with cetuximab for 9 months) and primary cetuximab-resistant SAS cells were investigated. The acquired resistance of UT5R9 cells to cetuximab was associated with increased proliferation activity, with a doubling time of 26 h compared with 45 h for the UT5 control cells (Fig. 1A). Cetuximab significantly inhibited the

Discussion

Acquired resistance to cetuximab has been reported to be associated with resistance to ionizing radiation [17] and to the EGFR-TK inhibitor gefitinib [22]. K-RAS mutations that result in the constitutive activation of K-Ras are one of the most important predictive markers for the lack of a tumor cell response to cetuximab, as shown for colorectal cancer [41], [43], [44]. In the current study, we showed that cetuximab radiosensitizes UT5 cells but not SAS cells. A similar result has been

Acknowledgments

The authors would like to thank Prof. Meier (Medical Clinic V, Research Group Molecular Oncology of Solid Tumors, Justus-Liebig-University Giessen, Germany) for providing the lonafarnib. We thank Dr. Wolfgang Eicheler, Dept. of Radiation Oncology, Technical University Dresden, for performing K-RAS sequencing. This work was supported by grants from the Deutsche Forschungsgemeinschaft [Ro527/5-1 (DFG-PAK190); SFB-773-TP B02] awarded to HPR and GRK 1302/2 (T11) awarded to MT/HPR.

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