Abstract 3003: The effects of autophagy inhibition on HNSCC sensitivity to CTX
Authors: Bradley Samantha T. , Lee Yong-Syu , Skiba Justin H. , Eckers Jaimee , Swick Adam D. , Hu Rong , Nickel Kwang P. , Gurel Zafer , Cosper Pippa F. , Kimple Randall J. 
Published: June 2022

Background: Cetuximab (CTX) is a monoclonal antibody commonly used in the treatment of head and neck squamous cell carcinomas (HNSCCs) that acts by targeting the epidermal growth factor receptor (EGFR). Addition of CTX to standard multi-agent chemotherapy only slightly improves the response rate from 20% to 36%, highlighting the urgent need for improved treatment. Autophagy is a naturally occurring mechanism activated within cells to protect them from harsh conditions or stress but can also provide cancerous cells with protection against further treatment attempts. Here, we examine the efficacy of combining CTX with the autophagy inhibitor, SAR405, as well as investigating the specific mechanism of CTX-induced autophagy.

Methods: To measure autophagy, we utilized the Promega LC3 reporter assay, Western blotting for LC3, immunofluorescence for LC3, and an acridine orange assay. We ran our assays using the HNSCC cell lines A253 (HPV-), UM-SCC1 (HPV+), and its CTX-resistant derivative, UM-SCC1-C5 (HPV+). Clonogenic assays were run to measure colony formation, cell counting kit 8 (CCK8) assays to determine cell survival, and flow cytometry to investigate cell cycle status.

Results: In this study, we first were able to demonstrate that CTX can activate pro-survival autophagy. CTX-treated cells had higher LC3 flux and more LC3 puncta than control cells as assessed by western blot and immunofluorescence staining, respectively. CTX-resistant cells demonstrated higher basal LC3 flux compared to the CTX-sensitive parental cells. We also discovered that the use of the autophagy inhibitor, SAR405, could help improve tumor response when combined with CTX. The addition of SAR405 to CTX treatment led to a decrease in colony formation and a decrease in cell survival, overcoming resistance to CTX. Interestingly, we discovered that SAR405 alone led to cell cycle arrest in the G1 phase but did not lead to a statistically significant decrease in cell survival compared to the control, showing that SAR405 is able to arrest cell growth but is not cytotoxic. Finally, knockdown of EGFR or LAPTM4B prevented CTX-induced activation of autophagy.

Conclusion: Through our work, we have been able to demonstrate that CTX is able to activate autophagy, and that this initiation of autophagy plays a cytoprotective role in HNSCC cells. The addition of the autophagy inhibitor SAR405 improved response to CTX treatment. The promising results obtained thus far open the door to future studies investigating the efficacy of combining CTX with more specific autophagy inhibitors.

Citation Format: Samantha T. Bradley, Yong-Syu Lee, Justin H. Skiba, Jaimee Eckers, Adam D. Swick, Rong Hu, Kwang P. Nickel, Zafer Gurel, Pippa F. Cosper, Randall J. Kimple. The effects of autophagy inhibition on HNSCC sensitivity to CTX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3003.