Human papillomavirus-driven repression of NRF2 signalling confers chemo-radio sensitivity and predicts prognosis in head and neck squamous cell carcinoma.

PURPOSE: To investigate the role of NRF2 signalling in conferring superior prognosis in patients with HPV positive (HPV+ve) head & neck squamous cell carcinomas (HNSCC) compared to HPV negative (HPV-ve) HNSCC and develop molecular markers for selection of HPV+ve HNSCC patients for treatment de-escalation trials. METHODS: NRF2 activity (NRF2, KEAP1, and NRF2-transcriptional targets), p16, and p53 levels between HPV+ve HNSCC and HPV-ve HNSCC in prospective and retrospective tumor samples as well as from TCGA database were compared. Cancer cells were transfected with HPV-E6/E7 plasmid to elucidate if HPV infection represses NRF2 activity and sensitizes to chemo-radiotherapy. RESULTS: Prospective analysis revealed a marked reduction in expression of NRF2, and its downstream genes in HPV+ve tumors compared to HPV-ve tumors. A retrospective analysis by IHC revealed significantly lower NQO1 in p16high tumors compared to p16low tumors and the NQO1 expression correlated negatively with p16 and positively with p53. Analysis of the TCGA database confirmed low constitutive NRF2 activity in HPV+ve HNSCC compared to HPV-ve HNSCC and revealed that HPV+ve HNSCC patients with 'low NQO1' expression showed better overall survival compared to HPV+ve HNSCC patients with 'high NQO1' expression. Ectopic expression of HPV-E6/E7 plasmid in various cancer cells repressed constitutive NRF2 activity, reduced total GSH, increased ROS levels, and sensitized the cancer cells to cisplatin and ionizing radiation. CONCLUSION: Low constitutive NRF2 activity contributes to better prognosis of HPV+ve HNSCC patients. Co-expression of p16high, NQO1low, and p53low could serve as a predictive biomarker for the selection of HPV + ve HNSCC patients for de-escalation trials.

Development of Moringa oleifera as functional food targeting NRF2 signaling: antioxidant and anti-inflammatory activity in experimental model systems.

Pharmacological activation of nuclear factor erythroid 2 related factor 2 (NRF2) provides protection against several environmental diseases by inhibiting oxidative and inflammatory injury. Besides high in protein and minerals, Moringa oleifera leaves contain several bioactive compounds, predominantly isothiocyanate moringin and polyphenols, which are potent inducers of NRF2. Hence, M. oleifera leaves represent a valuable food source that could be developed as a functional food for targeting NRF2 signaling. In the current study, we have developed a palatable M. oleifera leaf preparation (henceforth referred as ME-D) that showed reproducibly a high potential to activate NRF2. Treatment of BEAS-2B cells with ME-D significantly increased NRF2-regulated antioxidant genes (NQO1, HMOX1) and total GSH levels. In the presence of brusatol (a NRF2 inhibitor), ME-D-induced increase in NQO1 expression was significantly diminished. Pre-treatment of cells with ME-D mitigated reactive oxygen species, lipid peroxidation and cytotoxicity induced by pro-oxidants. Furthermore, ME-D pre-treatment markedly inhibited nitric oxide production, secretory IL-6 and TNF-α levels, and transcriptional expression of Nos2, Il-6, and Tnf-α in macrophages exposed to lipopolysaccharide. Biochemical profiling by LC-HRMS revealed glucomoringin, moringin, and several polyphenols in ME-D. Oral administration of ME-D significantly increased NRF2-regulated antioxidant genes in the small intestine, liver, and lungs. Lastly, prophylactic administration of ME-D significantly mitigated lung inflammation in mice exposed to particulate matter for 3-days or 3-months. In conclusion, we have developed a pharmacologically active standardized palatable preparation of M. oleifera leaves as a functional food to activate NRF2 signaling, which can be consumed as a beverage (hot soup) or freeze-dried powder for reducing the risk from environmental respiratory disease.