Abstract
Radiation resistance is a significant clinical problem in rectal cancer treatment, the mechanisms of which are poorly understood. NRF2 signalling is known to contribute to chemo/radioresistance in some cancers, but its role in therapeutic resistance in colorectal cancer (CRC) is unexplored. Using siRNA and CRiSPR/Cas9 isogenic CRC cell lines, we investigated the effect of the knockdown and upregulation of the NRF2 pathway on chemo-radiosensitivity. Poly (A) enriched RNA sequencing and geneset enrichment analysis (GSEA) were carried out on both sensitive and resistant cell models for mechanistic insights. Finally, a cohort of rectal patient samples was profiled to understand the clinical relevance of NRF2 signalling. Radioresistant cell lines were significantly radiosensitised by siRNA knockdown (SW1463, SER10 1.22, ANOVA p < 0.0001; HT55, SER10 1.17, ANOVA p < 0.01), but not the (already) radiosensitive HCT116. The constitutive activation of NRF2 via a CRISPR Cas9 NFE2L2 mutation, E79K, induced radioresistance in HCT116 (SER10 0.71, ANOVA, p < 0.0001). GSEA demonstrated significant opposing metabolic dependencies in NRF2 signalling, specifically, the downregulation of amino acid and protein synthesis with low levels of NRF2 and upregulation with over expression. In a clinical cohort of 127 rectal patients, using a validated mRNA signature, higher baseline NRF2 signalling was associated with incomplete responses to radiation higher final neoadjuvant rectal (NAR) score (OR 1.34, 95% C.I. 1.01–1.80, LRT p-value = 0.023), where high NAR indicates poor radiation response and poor long-term prognosis. This is the first demonstration of NRF2-mediated radiation resistance in colorectal cancer. NRF2 appears to regulate crucial metabolic pathways, which could be exploited for therapeutic interventions.
Highlights
Knockdown of NRF2 with RNAi resulted in a significant decrease in NRF2 signalling in three colorectal cancer (CRC) cell lines as assessed by validated NRF2 transcriptional targets, NQO1 and
Significant radiosensitisation was observed in NRF2-depleted SW1463
Compared to SW1463 and HT55, it had the lowest level of NRF2 activity (Supplementary Figure S1e)
Summary
Chemoradiation is the standard of care for all patients with locally advanced rectal cancer. Some patients respond poorly with little or no tumour regression [1], highlighting the need to better understand contributing factors. NRF2 is a potent transcription factor that plays a responsive role in cell protection against oxidative and electrophilic stress, as well as metabolic regulation. NRF2 is targeted for ubiquitination by KEAP1 resulting in its degradation. The constitutive activation of NRF2 signalling can occur by a number of distinct mechanisms, including somatic mutation of key regulators
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