PTH-272 Modulation of NRF2 alters the responsiveness of colorectal cancer cells to irinotecan

Abstract

<h3>Introduction</h3> Chemotherapy is essential to improve outcomes in colorectal cancer (CRC). Irinotecan is a pro-drug commonly used in CRC which requires conversion to the active metabolite Sn38. The cytoprotective protein NRF2 has been associated with chemo-resistance and a worse prognosis in a number of cancers.¹It also regulates the activity of enzymes (including CES1 and UGT1A1) that coordinate irinotecan metabolism and efflux. ²NRF2 remains bound to the regulatory protein KEAP1 in its quiescent state. The aim of this study is to establish the value of modulating NRF2 as a novel strategy for enhancing the efficacy of irinotecan-based chemotherapy either through increased conversion of irinotecan to Sn38 or by sensitisation of CRC cells to its effect. <h3>Method</h3> NRF2 was modulated in the human CRC cell line HCT116 using small inhibitory RNA (siRNA) targeting <i>NRF2</i>and <i>KEAP1 </i>or the chemical inhibitor brusatol and inducer CDDO-Me. Cells were then exposed to irinotecan over a range of concentrations for 48 h. Cell viability was measured by MTS assay and expressed as a percentage of control for the calculation of IC₅₀values. GraphPad Prism 6^®was used for all statistical analysis and the generation of dose-response curves. <h3>Results</h3> Successful NRF2 modulation was confirmed by western blotting for Nrf2 and downstream targets NQO1 and HO-1. The IC₅₀value of irinotecan in untreated control cells was 90µM. Induction of NRF2 by <i>KEAP1</i>siRNA (IC₅₀220µM) or CDDO-Me (IC₅₀325µM) significantly reduced irinotecan cytotoxicity (p &lt; 0.0001). In contrast, inhibition of NRF2 by <i>NRF2</i>siRNA (IC₅₀11µM) or brusatol (IC₅₀55µM) had the opposite effect (p &lt; 0.0001). <h3>Conclusion</h3> Inhibition of NRF2 sensitises CRC cells to irinotecan cytotoxicity and may allow improved response to treatment, or permit lower doses with fewer side effects. We are currently validating these findings <i><i>in vivo</i></i>and examining the contribution of processes that mediate the disposition of irinotecan. The availability of NRF2 inhibiting and inducing compounds may allow for successful modulation in the clinical setting. <h3>Disclosure of interest</h3> J. Evans Grant/ Research Support from: Cancer Research UK, B. Winiarski: None Declared, P. Sutton: None Declared, D. Palmer: None Declared, N. Kitteringham: None Declared. <h3>References</h3> Kawasaki Y, Ishigami S, Arigami T, Uenosono Y, Yanagita S, Uchikado Y, <i>et al</i>. Clinicopathological significance of nuclear factor (erythroid-2)-related factor 2 (Nrf2) expression in gastric cancer. BMC Cancer 2015;<b>15</b>(1):5 Wang M, Zhu JY, Chen S, Qing Y, Wu D, Lin YM, <i>et al</i>. Effects of co-treatment with sulforaphane and autophagy modulators on uridine 5’-diphospho-glucuronosyltransferase 1A isoforms and cytochrome P450 3A4 expression in Caco-2 human colon cancer cells. Oncol Lett. 2014;8(6):2407–16