Association of NRF2 with HIF-2?-induced cancer stem cell phenotypes in chronic hypoxic condition

The acquisition of the cancer stem cell (CSC) properties is often mediated by the surrounding microenvironment, and tumor hypoxia is considered an important factor for CSC phenotype development. High levels of NRF2 (Nuclear Factor Erythroid 2-Like 2; NFE2L2), a transcription factor that maintains cellular redox balance, have been associated with facilitated tumor growth and therapy resistance. In this study, we investigated the role of NRF2 in hypoxia-induced CSC phenotypes in colorectal cancer cells. Chronic hypoxia for 72 h resulted in CSC phenotypes, including elevation of krupple-like factor 4 (KLF4) and octamer-binding transcription factor 4 (OCT4), and an increase in cancer migration and spheroid growth with concomitant hypoxia-inducible factor 2 alpha (HIF-2 alpha) accumulation. All these chronic hypoxia-induced CSC properties were attenuated following HIF-2 alpha-specific silencing. In this chronic hypoxia model, NRF2 inhibition by shRNA-based silencing or brusatol treat-ment blocked HIF-2 alpha accumulation, which consequently resulted in decreased CSC marker expression and in-hibition of CSC properties such as spheroid growth. In contrast, NRF2 overactivation by genetic or chemical approach enhanced the chronic hypoxia-induced HIF-2 alpha accumulation and cancer migration. As a molecular mechanism of the NRF2-inhibition-mediated HIF-2 alpha dysregulation, we demonstrated that miR-181a-2-3p, whose expression is elevated in NRF2-silenced cells, targeted the HIF-2 alpha 3 ' UTR and subsequently suppressed the chronic hypoxia-induced HIF-2 alpha and CSC phenotypes. The miR-181a-2-3p inhibitor treatment in NRF2-silenced cells could restore the levels of HIF-2 alpha and CSC markers, and increased cancer migration and sphere formation under chronic hypoxia. In line with this, the miR-181a-2-3p inhibitor transfection could increase tumorigenicity of NRF2-silenced colorectal cancer cells. Collectively, our study suggests the involvement of NRF2/miR181a-2-3p signaling in the development of HIF-2 alpha-mediated CSC phenotypes in sustained hypoxic environments.