Abstract 3053: Inhibition of heregulin-mediated ErbB3 signaling as a radiosensitization therapy for head and neck cancers

Abstract

Abstract PURPOSE: EGFR signaling confers resistance to radiation therapy (RT) and is a validated target in head and neck squamous cell carcinoma (HNSCC). Inhibition of EGFR function in combination with RT improves local control and overall survival in this patient population, however, the mechanisms of resistance to combined treatment with RT + cetuximab are incompletely understood. We sought to develop cell line models of true cetuximab resistance and to define the molecular characteristics of these tumor cells with respect to radiation sensitivity. EXPERIMENTAL PROCEDURES: A431 and FaDu cell lines with resistance to cetuximab were generated by two methods: (i) exposure to increasing doses of cetuximab over 14 weeks or (ii) single cell clonal selection prior to identical cetuximab exposure regimens or for untreated controls. Receptor tyrosine kinase activity and dependent downstream signaling were assessed by phospho-blot analysis. Proliferation was determined with MTT. Radiosensitivity was determined through clonogenic survival analysis. The EFM-19 cell line was used as a functional indicator of heregulin family ligand expression. The efficacy of cetuximab was assessed in xenografts grown in nude mice. RESULTS: Cell line cultures exposed to increasing doses of cetuximab achieved only partial resistance in vivo. The process of clonal selection allowed us to identify distinct cell populations: (a) cetuximab-sensitive clones from untreated controls, (b) partial-cetuximab resistant clones from untreated controls, and (c) cetuximab-resistant clones from cell lines exposed to increasing doses. Partial- and cetuximab-resistant clones showed an increase of phospho-ErbB3, AKT, and Met. These clones were also more resistant to ionizing radiation. We also tested whether KTN3379, a monoclonal antibody that inhibits ligand-dependent and independent ErbB3 activation, affected cell proliferation and survival. After 5 days, KTN3379 + cetuximab reduced proliferation of cetuximab-resistant clones but did not further blocked partial- and sensitive-clones compared to cetuximab alone. This corresponded to a significant reduction of ErbB3/Akt (and Met) mediated by KTN3379. Using the EFM-19 cell model, which lacks endogenous heregulin, we demonstrate that cetuximab resistant cells upregulate heregulin family ligand expression and that KTN3379 effectively blocks this autocrine survival signaling. CONCLUSION: Tumor cell heterogeneity obscures the identification of operative resistance mechanisms in vitro. Autocrine ligand activation of ErbB3 is a mechanism for therapeutic resistance to cetuximab and may also cause radioresistance. Cross-resistance to both therapeutic modalities identifies HRG and ErbB3 as attractive therapeutic targets for improving delivery of radiation therapy for HNSCC. Citation Format: Marta Baro, Josep Balart, Joseph N. Contessa. Inhibition of heregulin-mediated ErbB3 signaling as a radiosensitization therapy for head and neck cancers. [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 3053.