Oxidative stress associated metabolic adaptations regulate radioresistance in human lung cancer cells

Differential inherent and acquired radioresistance of human lung cancer cells contribute to poor therapeutic outcome and tumor recurrence after radiotherapy. Inherent radioresistance of lung cancer cells is known to be associated with ROSLow cancer stem cells (CSCs). However, mechanism of acquired radioresistance in lung cancer cells is poorly understood. Here, we exposed human lung cancer cells (A549) to a cumulative dose of 40Gy and allowed the radioresistant (RR) survivors to divide and form macroscopic colonies after each fraction of 5Gy dose. The RR subline exhibited enrichment of cytosolic ROSHigh cells without specific increase in mitochondrial ROS levels. We found a concomitant increase in the expression of redox regulatory transcription factor Nrf2 and its dependent antioxidant genes in RR cells and cell cycle delay as compared to parental cells. The treatment of RR cells with Nrf2 inhibitor resulted in decreased clonogenic survival indicating their addiction to Nrf2 for metabolic adaptations under high levels of cytosolic ROS. A causal role of inherent ROS levels in conferring radioresistance was established by sorting ROSHigh and ROSLow populations from parental and RR cells. It was observed that ROSHigh population from both parental and RR cells exhibited radioresistance as observed by clonogenic assay. Interestingly, ROSHigh population of cells exhibited higher levels of cellular thiols in both parental and RR cells. Thus, our observations highlight presence of a novel subpopulation in lung cancer cells, which exhibits radioresistance by maintaining 'oxidative stress' and Nrf2 dependent metabolic adaptations. We also posit Nrf2 pathway as a druggable target for radiosensitization of RR A549 cells.