Introduction: Head and neck squamous cell carcinomas (HNSCCs) afflict over half a million patients annually worldwide. In the absence of disease at distant sites, salvage treatment may provide durable disease control in only ∼15% of such patients. Our research goal is to improve radiotherapy for aggressive HNSCCs by identifying novel targets for radiosensitization. The EphB4 receptor is ubiquitously expressed in HNSCCs and has been shown to promote tumorigenic and invasive properties of HNSCCs but the effect of EphB4 on cellular radiosensitization has not been investigated. We hypothesize that knockdown of EphB4 receptor will enhance radiosensitization of HNSCCs by inhibiting EphB4 targets involved in radioresistance.

Materials/Methods: To determine the in vitro radiosensitization effect following EphB4 knockdown, we used EphB4-targeting siRNA in clonogenic assays in HNSCC cell lines. Effects of EphB4-siRNA on cell cycle progression, DNA damage response, and cell death pathways were also investigated. For in-vivo testing, an EphB4 blocking protein was used to investigate the radiosensitization in a patient derived xenograft (PDX) model of HNSCC.

Results: We observed a decrease in the survival fractions in HNSCC cell lines following knockdown of EphB4 at increasing doses of radiation. Cell cycle analysis showed an enhanced G2 arrest of HNSCC cells following EphB4 knockdown and radiation exposure. We also observed an increase in the expression of p-H2AX, a DNA damage marker protein, in HNSCC cells suggesting activation of DNA damage response pathway following EphB4 knockdown and radiation exposure. This was further accompanied by enhanced percentage of apoptotic cells as evident in TUNEL assay and modulation of key apoptotic markers. Several pro-survival markers, including, p-AKT, p-EGFR, EGFR, and Bcl-XL were markedly decreased in the EphB4-knockdown and radiation combination group. Data using an in-vivo PDX animal model of HNSCC showed reduction in tumor growth following administration with an optimal dose of EphB4 blocking protein or XRT alone. This reduction in tumor growth was significantly augmented when EphB4 blocking protein was administered in combination with radiation demonstrating radiosensitization effect in an in vivo PDX model of HNSCC.

Conclusions: Our findings support the hypothesis that EphB4 promotes resistance of HNSCCs to ionizing radiation and its targeted inhibition will therefore result in enhanced radiosensitization both in vitro and in vivo. This data should serve as the basis for a clinical trial design for combination therapy of anti-EphB4 with radiation in patients with locally advanced head and neck cancer.

Citation Format: Shilpa Bhatia, Kellen Hirsch, Jaspreet Sharma, Stephen Keysar, Antonio Jimeno, Parkash Gill, Xiao-Jing Wang, Sana D. Karam. Role of EphB4 in radiosensitization of head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-127.