Abstract 2946: Role of EphB3 receptor in mediating head and neck tumor growth, cell migration, and response to PI3K inhibitor
Authors: Bhatia Shilpa , Griego Anastacia , Lennon Shelby , Oweida Ayman , Sharma Jaspreet , Rohmer Christina , Uyanga Nomin , Bukkapatnam Sanjana , Court Benjamin Van , Raben David , Young Christian , Heasley Lynn , Karam Sana D. 
Published: August 2018

Introduction: Eph proteins have emerged as critical drivers affecting tumor growth and progression in human malignancies. Dysregulated expression of EphB3, a member of the Eph receptor gene family, has been reported in different cancers. However, the precise role of EphB3 in head and neck squamous cell carcinoma (HNSCC) has not been explored. In the current study, we examined the effect of EphB3 downregulation on HNSCC tumor cell growth, migration, and response to PIK3CA inhibition both in vitro and in an orthotopic model of head and neck cancer.

Materials and methods: Alterations in EPHB3 across cancer types in the TCGA database were accessed via cBioportal. shRNA approach was used to knockdown the levels of EphB3 in human and murine HNSCC cell lines. The effect of EphB3 knockdown on cell viability and cell migration was determined by MTT/Trypan blue assay and by Boyden chamber assay respectively. We also generated a BKM120 resistant HNSCC cell line to elucidate the functional role of EphB3 in mediating sensitivity to PI3K inhibitor. The phospho-EphB3 levels were analyzed by an immunoprecipitation assay. For in vivo testing, we used an orthotopic model of HNSCC. Immunohistochemistry and Western blot analysis was performed on cultured HNSCC cells and on tumor tissues to determine key proteins that are altered in response to EphB3 knockdown in the presence and absence of PI3K inhibition.

Results: Our TCGA data analysis showed that EphB3 is frequently co-amplified with PIK3CA in HNSCC. We therefore hypothesized that EphB3 amplification plays a pro-tumorigenic role in HNSCC and that EphB3 and PIK3CA are co-operating oncogenes that contribute toward its pathogenesis. This hypothesis was not experimentally supported since EphB3 knockdown failed to alter HNSCC tumor cell growth in vitro or in vivo with an orthotopic model. However, responsiveness of EphB3 knockdown tumors to the PI3K inhibitor, BKM120, was significantly decreased in terms of both tumor growth delay and survival. This is associated with an increase in pro-survival proteins, S6 and BcL-XL in the EphB3 shRNA tumors treated with BKM120 compared to controls. We further observed that EphB3 knockdown resulted in increased migration in vitro and increased EMT gene signature in vivo. To explain these results, we examined EphB3 phosphorylation levels in HNSCC at baseline. While total EphB3 levels were high, we found low phospho-EphB3 levels in HNSCCs. Forced EphB3 phosphorylation with an ephrin-B2-Fc fusion protein resulted in decreased HNSCC migration and cell growth and enhanced response to BKM120 in vitro.

Conclusions: Our data collectively indicate that progression of HNSCC selects for low/inhibited EphB3 activity to enhance their survival and migratory abilities and decrease response to PI3K inhibitor. Therefore, strategies focused on activating EphB3 might be helpful to inhibit tumor growth and enhance sensitivity to PI3K inhibitors in HNSCC.

Citation Format: Shilpa Bhatia, Anastacia Griego, Shelby Lennon, Ayman Oweida, Jaspreet Sharma, Christina Rohmer, Nomin Uyanga, Sanjana Bukkapatnam, Benjamin Van Court, David Raben, Christian Young, Lynn Heasley, Sana D. Karam. Role of EphB3 receptor in mediating head and neck tumor growth, cell migration, and response to PI3K inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2946.