Nrf2/ASPM axis regulated vasculogenic mimicry formation in hepatocellular carcinoma under hypoxia

Hypoxic microenvironment is a common feature of most solid tumors including hepatocellular carcinoma (HCC). Vasculogenic mimicry (VM) formation by tumor cells could provide blood supply to tumor cells under hypoxia. NFE2 like basic leucine zipper (bZIP) transcription factor 2 (Nrf2), a regulator of cellular homeostasis, may promote tumor progression in the hypoxic conditions. However, the role and regulatory mechanisms of Nrf2 in HCC are not fully elucidated.

Nrf2 and assembly factor for spindle microtubules (ASPM) expression modulations were conducted by lentiviral transfections. Western blot, immunofluorescence, ChIP-qPCR, dual-luciferase reporter gene assay, flow cytometry, RNA sequencing, multiple bioinformatics databases analysis, cell function assays in vitro, mouse model in vivo and human HCC tissues were employed to assess the effect of Nrf2/ASPM axis on HCC progression under hypoxia.

Nrf2 and ASPM expression facilitated epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) feature, and VM formation of HCC cells under hypoxia. Furthermore, Nrf2-regulated ASPM expression, via binding directly to the promoter region of ASPM and transcriptionally promoting ASPM expression. ASPM re-expression in Nrf2 knockdown cells or ASPM knockdown in Nrf2 overexpression cells reversed the cellular function caused by Nrf2. Meantime, retinol metabolism pathway was disrupted following abnormal ASPM expression. Nrf2/ASPM axis in murine models accelerated tumor growth and VM, corroborating in vitro findings. All-trans retinoic acid treatment reversed stemness and VM of HCC cells in vitro and in vivo. Clinically, Nrf2 and ASPM expressions were related to poor prognosis of HCC patients.

Nrf2 drives EMT, CSCs characteristics and VM in HCC under hypoxia through the modulation of ASPM. Retinol metabolism pathway was dysregulated in HCC cells with ASPM overexpression. Nrf2/ASPM axis and related pathway provided potential therapeutic target for HCC.