Abstract 1344: Autophagy contributes to therapeutic resistance in head and neck cancer

Abstract

Abstract Background: In the curative setting for head and neck cancer (HNC) a common treatment is radiation combined with cetuximab, an antibody therapeutic targeting EGFR. Despite decades of research into improved treatments, therapeutic resistance remains a major challenge for this malignancy, with roughly 40% of patients developing recurrent disease. Recent evidence has suggested that autophagy, a cellular stress response, may be an additional contributor to therapy resistance, by protecting HNC cells from the cytotoxic effects of radiotherapy and the growth inhibitory effects of anti-EGFR treatment. The mechanism of radiation-induced autophagy is under current investigation. Methods: Cell lines were source from commercial sources, cultured under recommended conditions, and identity confirmed by short tandem repeat testing. Induction of autophagy was detected by immunoblot flux assays for LC3 and p62, immunofluorescent staining of autophagic vesicles, LC3 reporter flux assay, and flow cytometry using acridine orange. The effect of autophagy inhibition was tested using clonogenic survival assays. Induction of apoptosis was analyzed by immunoblot against cleaved caspase and PARP and via AnnexinV staining. Results: We evaluated a panel of both human papillomavirus (HPV) positive and negative HNC cell lines for autophagic response to both cetuximab (CTX) treatment and ionizing radiation (XRT). Flux assays revealed that both CTX and XRT treatment induced autophagy in a time- and dose-dependent manner. Immunofluorescent staining of LC3 to identify autophagic vesicles showed that a relatively small fraction of the total cell population is able to induce this response. Flow cytometry analysis demonstrated that autophagic cells were largely non-apoptoic. For example, in the UM-SCC47 cell line treated with CTX for 48 h, flow cytometry for autophagy (20.8%), apoptosis (13.7%) or dual staining (5.4%) suggests a cytoprotective role for autophagy. The addition of the ULK1 inhibitor, SBI-0206965) to CTX and XRT induced apoptosis as shown by caspase activity and AnnexinV staining and reduced clonogenic cell survival. Conclusions: These preclinical studies have established the proof of concept for the cytoprotective effect of autophagy in response to anticancer treatments including EGFR inhibition and radiotherapy in HNC. Further, we have identified the addition of specific autophagy inhibitors to standard treatments as a potential strategy to overcome this mechanism of resistance. Citation Format: Jaimee Eckers, Justin Skiba, Gopika Senthilkumar, Kwang P. Nickel, Adam D. Swick, Randall J. Kimple. Autophagy contributes to therapeutic resistance in head and neck cancer [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 1344.