Risk stratification in rectal cancer: identifying tumours resistant to pre-operative radiotherapy

Abstract

Substantial progress has been made in the last few decades in the therapeutic and surgical treatment of CRC designed to improve survival. However, treatments tailored to the individual patient based on the mutation and gene expression profiles of CRC remain elusive. Rectal cancer, in particular, has come under scrutiny because of the wide variation in response rates to neoadjuvant radiotherapy, which may be linked to the high degree of hypoxia observed in this cancer. The cell killing potential of radiation is determined by the presence of oxygen and reactive oxygen species (ROS). ROS can be induced during inflammation and may cause genetic damage and cellular transformation, aiding radiation-induced DNA damage. Dual oxidase 2 (DUOX2) is a major source of ROS in the intestine and this study aims to evaluate the expression of DUOX2 and its maturation factor (DUOXA2) in both normal and neoplastic tissue, including rectal cancer, to determine whether oxygen tension can influence gene expression and to measure the outcome of exposure to irradiation (IR) in rectal cancer cell lines expressing different levels of DUOX2/DUOXA2. MicroRNAs (miRNAs) were also investigated as potential markers of hypoxia in rectal cancer cell lines and tissue. DUOX2 and DUOXA2 expression was found to be low in normal mucosal tissue, highly expressed in adenomas and moderately expressed in cancers, indicating a potential link between elevated ROS levels and tumour progression. Rectal cancer cell lines were used to show in vitro that DUOX2 expression was upregulated in response to a reduction in oxygen tension (2 % O2) and that the alteration in DUOX2 expression was independent of COX2 and HIF-1α expression. With the addition of IR, cells with high DUOX2 expression showed high levels of DNA damage and low numbers of stem-like cells (CD24) indicating susceptibility to IR. Cells with low DUOX2 expression showed low levels of DNA damage, high survival rate and a high quantity of CD24+ cells indicating resistance to IR. A number of consistently upregulated miRNAs were also identified in cells lines maintained in hypoxia. Follow-up investigations showed miR-210 to be consistently upregulated in hypoxic regions of rectal cancer and to target the iron sulphur cluster homolog (ISCU) gene both in vitro and in vivo. ISCU is an essential part of the mitochondrial electron transport chain and can control ROS production. Both DUOX2 and miR-210 have significant promise as markers of hypoxia in rectal cancers and warrant further investigation as markers of response to neoadjuvant radiotherapy.