Overactivated NRF2 induces pseudohypoxia in hepatocellular carcinoma by stabilizing HIF-1 alpha

Abstract

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is highly expressed/activated in most hypoxic tumors including hepato-cellular carcinoma (HCC). Another key transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2), is also constitutively overactivated in HCC. In an attempt to determine whether HIF-1 alpha and NRF2 could play complementary roles in HCC growth and progression, we investigated the crosstalk between these two tran-scription factors and underlying molecular mechanisms in cultured HCC cells and experimentally induced hepatocarcinogenesis as well as clinical settings. While silencing of HIF-1 alpha in HepG2 human hepatoma cells did not alter the protein expression of NRF2, NRF2 knockdown markedly reduced the nuclear accumulation of HIF-1 alpha without influencing its mRNA expression. In diethylnitrosamine-induced hepatocarcinogenesis in wild type mice, there was elevated NRF2 expression with concomitant upregulation of HIF-1 alpha. However, this was abol-ished in Nrf2 knockout mice. NRF2 and HIF-1 alpha co-localized and physically interacted with each other as assessed by in situ proximity ligation and immunoprecipitation assays. In addition, the interaction between NRF2 and HIF-1 alpha as well as their overexpression was found in tumor specimens obtained from HCC patients. In normoxia, HIF-1 alpha undergoes hydroxylation by a specific HIF-prolyl hydroxylase domain protein (PHD), which facilitates ubiquitination and proteasomal degradation of HIF-1 alpha. NRF2 contributes to pseudohypoxia, by directly binding to the oxygen-dependent degradation (ODD) domain of HIF-1 alpha, which hampers the PHD2-mediated hydroxyl-ation, concomitant recruitment of von-Hippel-Lindau and ubiquitination of HIF-1 alpha.