In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone – a steroid hormone that regulates developmental transitions in insects. We use Drosophila melanogaster as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition – a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.

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COPurpose/Objective(s): 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 approximately 15% of such patients. Our research goal is to improve radiation therapy 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 fulfill our objective, we used EphB4-targeting siRNA and performed clonogenic assays in HNSCC cell lines to determine the in vitro radiosensitization effect following EphB4 knockdown. Effects of EphB4-siRNA on cell cycle progression, DNA damage response, and cell death pathways were also investigated. 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. In addition, we 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 DNA fragmentation and modulation of key apoptotic markers. Studies are currently underway to determine the effect of EphB4 inhibition on radiosensitization in an in vivo patient-derived xenograft model of HNSCC. Conclusion: Our findings support the hypothesis that EphB4 promotes resistance of HNSCCs to ionizing radiation and its targeted inhibition will therefore result in enhanced radiosensitization. In conclusion, the successful completion of this study will allow us to design functional analyses of EphB4 targets responsible for radioresistance. From translational point of view, these targets could serve as the basis for development of combined therapy, prognostic biomarkers, or patient selection strategies in future clinical trials.