Abstract PO-074: Molecular signatures of radioresistance in radioresistant cancer cell lines
Authors: Yeo Eugenia L.L. , Chu Pek Lim , Poon Dennis J.J. , Susanti Dewi , Sim Adelene Y.L. , Chua Melvin L.K. 
Published: April 2021

Abstract
Radiotherapy represents a main treatment modality for cancers. However, a subset of tumors manifest resistance to radiotherapy. Mechanistically, this is opined to occur via selection of de novo radioresistant (RR) clones and subsequent adaptation leading to an aggressive tumor phenotype. To better understand the molecular processes underpinning this disease model, here, we performed whole transcriptome profiling (Illumina TruSeq, CA) of three RR cancer cell lines – 22Rv1 prostate, FaDu hypopharyngeal and C666-1 nasopharyngeal cancer induced by exposure to a prolonged course of fractionated radiotherapy (2 Gy x 45 fr, 0.66 MeV, 0.716 Gy/min). Differential expression analysis was performed with pairwise comparisons of our RR versus WT lines. Variant calling using RR-WT cell line pairs was performed using three different variant calling tools (mutect2, strelka2, verdict) to determine mutations that may potentially drive radioresistance. In doing so, we attempt to elucidate transcriptomic signatures of radioresistance that may be consistent across different cancers where RT is a main treatment modality. Radioresistance of our RR cell lines was confirmed by clonogenic survival (surviving fraction ratio at 2 Gy [SF2Gy,RR/SF2Gy,WT] ranged from 1.08 [FaDu] to 1.40 [22Rv1]). We found significant upregulation of genes (adjusted p-value &lt;0.1 and log2 fold change &gt;1) involved in tissue remodeling (MMP9/10, PRSS2/23), proliferation (FAM13C), invasion (CNTN1, FGF18, PROM1) and stemness (ATF3, CEMIP). We observed downregulation of tumor suppressive genes (ARHGAP6, BEX1). Notably, we found more differentially expressed genes (DEGs) associated with radioresistance than previously reported. Additionally, while some of these previously reported DEGs were separately investigated in specific cancers (ATF3, BEX1, IL1RL1, MMP10), here they were found consistently in three cancer types. Next, gene-set enrichment analyses highlighted upregulation of key pathways such as hypoxia and epithelial-mesenchymal transition. However, there were no variants common among the three RR cell lines detected. Next, we probed the DNA damage response of WT and RR lines to irradiation as a function of time post-irradiation (1-24 h) and radiation dose (0-4 Gy) in the 22Rv1 prostate cancer model. We observed more rapid and greater upregulation of DNA damage response (ATM, Rad51, DNA-PKcs) in RR- relative to WT-22Rv1. We also found elevated pro-survival and tissue remodeling signaling in RR- relative to WT-22Rv1, suggesting that these pathways are elevated both at baseline and in response to RT-induced stress. Herein, by standardizing the fractionation scheme for generating RR sublines, we compared dysregulated genes and pathways that appear to drive the RR phenotype across three different cell lines. Interestingly, consistent with our results, data from the human protein atlas suggests that upregulation of PRSS2/23 is negatively associated with patient survival in head and neck cancers, highlighting the potential relevance of our work in the clinic.
Citation Format: Eugenia L.L. Yeo, Pek Lim Chu, Dennis J.J. Poon, Dewi Susanti, Adelene Y.L. Sim, Melvin L.K. Chua. Molecular signatures of radioresistance in radioresistant cancer cell lines [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-074.