Abstract 4431: TRAIL resistance by radiation induced hypermethylation of DR4 CpG island in recurrent laryngeal squamous cell carcinoma .
Authors: Lee Jong Cheol , Lee Won Hyeok , Min Young Joo , Lee Yoon-Se , Park Jung Je , Kim Seong Who , Kim Sang Yoon 
Published: August 2014

Abstract
The radiotherapy has been known as one of standard treatment methods in larynx cancer, and the many targeted therapies have been developed for improving radiotherapy. In this regard, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be one promising candidate, because TRAIL has a high selectivity for tumor cells and radiation efficiently sensitizes tumor cells towards apoptosis induction by TRAIL. It has been showing that irradiation strongly sensitizes malignant cells to TRAIL-induced apoptosis without any synergistic effects in normal tissues. Up to now, however, most studies have focused on immediate
TRAIL-effect after small amount of irradiation. In this study, we aim to investigate the effect of chronic irradiation on TRAIL response to see if TRAIL could be an effective targeted therapy in recurrent laryngeal squamous cell carcinoma.
Using previously established AMC-HN3 cell line from laryngeal SCCa patient, we have generated isogenic model of chronic irradiated HN3R cell line after every 2 or 3 day irradiation of 2 Gy/day of X-rays (70 Gy cumulative doses). Interestingly, through MTS assay, HN3R cells show much more resistance to TRAIL induced apoptosis than HN3 cells. Among suggested mechanisms, we found that a significantly reduced DR4 receptor is the cause of this resistance. Reverse transcription-PCR, western blotting and FACS analysis for DR4 receptors show that the expression of mRNA and the total amount, and surface expression of DR4 protein was much less in HN3R cells. Inhibition of DR4 by antagonistic antibody or si-DR4 significantly decreased TRAIL sensitivity in HN3 cells, and overexpression of DR4 enhanced the TRAIL sensitivity in HN3R cells. To evaluate this result in laryngeal cancer specimens, tissue microarray with 81 advanced laryngeal cancer specimens was constructed. Immunohistochemical assessment score of DR4 staining in 23 salvage surgical specimens after radiation failure shows statistically significant lower score than 58 surgical specimens without radiation treatment (3.09±2.23 vs.
4.43±3.36, p=0.040). Postulating that
DR4 gene might be silenced by hypermethylation after long-term irradiation, methylation-specific PCR for DR4 was undertaken. Compared to HN3 cells, HN3R cells had a methylated 5’ regulatory CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2’-deoxycytidine (5-AZAdC). In conclusion, chronic irradiation induces the TRAIL resistance by hypermethylation of CpG island of DR4 receptor in recurrent laryngeal squamous cell carcinoma. Thus, the status of receptor methylation might be one of biomarker to predict TRAIL response especially after radiation failure.
Citation Format: Jong Cheol Lee, Won Hyeok Lee, Young Joo Min, Yoon-Se Lee, Jung Je Park, Seong Who Kim, Sang Yoon Kim. TRAIL resistance by radiation induced hypermethylation of DR4 CpG island in recurrent laryngeal squamous cell carcinoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4431. doi:10.1158/1538-7445.AM2013-4431