Elsevier

Radiotherapy and Oncology

Volume 105, Issue 2, November 2012, Pages 241-249
Radiotherapy and Oncology

Molecular radiobiology
Dasatinib, a multi-kinase inhibitor increased radiation sensitivity by interfering with nuclear localization of epidermal growth factor receptor and by blocking DNA repair pathways

https://doi.org/10.1016/j.radonc.2012.08.010Get rights and content

Abstract

Background and purpose

Although inhibition of epidermal growth factor receptor (EGFR) signaling during radiation led to improvement of tumor control and survival, novel strategies are needed to further improve the outcome of patients with locally advanced head and neck carcinoma. Because EGFR is known to interact with c-Src kinases, the present study investigated dasatinib (BMS-354825), an inhibitor of c-Src kinases, for its efficacy in enhancing radiosensitivity of human head and neck squamous cell carcinomas (HNSCC) in vitro and examined the underlying mechanisms for this effect.

Materials and methods

Six HNSCC lines were exposed to dasatinib, radiation, or both, and assessed for c-Src and EGFR expression, cell survival and colony forming ability. Among these cell lines, HN-5 and FaDu lines were analyzed for induction of apoptosis, cell cycle re-distribution and for nuclear localization of EGFR, γ-H2AX and 53BP1 proteins. Immuno-precipitation and Western blots were performed to analyze the levels and binding of proteins involved in cell survival, apoptosis and DNA repair pathways. Suppression of c-Src by siRNA and subsequent clonogenic assay was performed in HN-5 cells.

Results

All six HNSCC lines that were examined expressed high levels of c-Src. Two (HN-5 and MDA-183) expressed higher levels of EGFR than other lines. Dasatinib suppressed cell survival of all cell lines tested independent of c-Src or EGFR levels but enhanced the radiosensitivity of HN-5 and MDA-183. HN-5 and FaDu were analyzed further. Dasatinib suppressed phosphorylation of c-Src in both cell lines, but decreased repair of radiation-induced DNA damage in HN-5 cells only as evidenced by suppression of c-Abl and Nbs-1 activity, inhibition of the association between c-Src and EGFR or Her-2, prolongation of nuclear γ-H2AX and 53BP1 foci and inhibition of EGFR nuclear localization and its association with DNA-PKcs. Finally, partial suppression of c-Src resulted in a small increase in HN-5 cell radiosensitivity.

Conclusions

Our data demonstrate that dasatinib induces apoptosis and blocks DNA repair in EGFR-expressing HNSCC cells and improves radiotherapy outcome. These findings warrant further investigation using in vivo tumor models for potential translation into clinical testing.

Section snippets

Materials

Dasatinib was obtained from Bristol-Myers Squibb (New York, NY). Primary antibodies, EGFR, Her-2, PARP and tubulin were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). All other primary antibodies were from Cell Signaling Technology (Beverly, MA). Secondary antibodies conjugated with horse-radish peroxidase and ECL-plus were from GE Healthcare (Piscataway, NJ). Fluorescent probes, DAPI was from Sigma Chemical Company (St. Louis, MO), YO-PRO1 was from Invitrogen Life Technologies (Carlsbad,

Basal levels of c-Src and EGFR in HNSCCs

Whole cell lysates were processed for Western blot analysis and expression levels of c-Src and EGFR were quantified. Fig. 1A shows that all HNSCCs constitutively expressed c-Src protein. However, EGFR expression levels were higher in HN-5 and MDA-183 than the other four cell lines. Assessment of cell viability by MTT assay showed that dasatinib (5–200 nM, 72 h) exerted differential effect on the six lines tested. The IC50 values for four cell lines (HN-5, UMSCC-1, MDA-183 and UMSCC-22A) ranged

Discussion

The present study demonstrated that dasatinib has the potential to increase tumor cell response to radiotherapy in some tumors but not in others. Our study suggests three ways in which c-Src and its inhibition may differentially regulate cellular viability and radiation sensitivity.

First, HNSCCs may possess alternative pathways for survival. Dasatinib suppressed phosphorylation of its target molecule, c-Src and FAK, a protein that is associated with c-Src activity in HN-5 and FaDu cell lines

Conflict of Interest Statement

Dr. K.K. Ang served on Advisory Board of Bristol-Myers Squibb Company. All other authors have no conflict of interest.

Acknowledgments

This study was supported by a sponsored agreement with Bristol-Myers Squibb Company (U.R.), Multidisciplinary Research Program at University of Texas M.D. Anderson Cancer Center (B.G.), DAMD17-01-1-0689 05, DOD US Army (B.G.), NIH PO-1 Grant CA-06294 (K.K.A.), Gilbert H. Fletcher Memorial Distinguished Chair (K.K.A.) and a UICC ACSBI fellowship supplemented by University of Zurich (O.R.).

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    Current address: Department of Radiation Oncology, University Hospital Zurich, Ramistrasse 100, 8091 Zurich, Switzerland.

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