BackgroundCycling and chronic tumor hypoxia are involved in tumor development and growth. However, the impact of cycling hypoxia and its molecular mechanism on glioblastoma multiforme (GBM) progression remain unclear.MethodologyGlioblastoma cell lines, GBM8401 and U87, and their xenografts were exposed to cycling hypoxic stress in vitro and in vivo. Reactive oxygen species (ROS) production in glioblastoma cells and xenografts was assayed by in vitro ROS analysis and in vivo molecular imaging studies. NADPH oxidase subunit 4 (Nox4) RNAi-knockdown technology was utilized to study the role of Nox4 in cycling hypoxia-mediated ROS production and tumor progression. Furthermore, glioblastoma cells were stably transfected with a retroviral vector bearing a dual reporter gene cassette that allowed for dynamic monitoring of HIF-1 signal transduction and tumor cell growth in vitro and in vivo, using optical and nuclear imaging. Tempol, an antioxidant compound, was used to investigate the impact of ROS on cycling hypoxia-mediated HIF-1 activation and tumor progression.Principal FindingsGlioblastoma cells and xenografts were compared under cycling hypoxic and normoxic conditions; upregulation of NOX4 expression and ROS levels were observed under cycling hypoxia in glioblastoma cells and xenografts, concomitant with increased tumor cell growth in vitro and in vivo. However, knockdown of Nox4 inhibited these effects. Moreover, in vivo molecular imaging studies demonstrated that Tempol is a good antioxidant compound for inhibiting cycling hypoxia-mediated ROS production, HIF-1 activation, and tumor growth. Immunofluorescence imaging and flow cytometric analysis for NOX4, HIF-1 activation, and Hoechst 3342 in glioblastoma also revealed high localized NOX4 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion within the endogenous solid tumor microenvironment.ConclusionsCycling hypoxia-induced ROS via Nox4 is a critical aspect of cancer biology to consider for therapeutic targeting of cycling hypoxia-promoted HIF-1 activation and tumor progression in GBM.

AXL which is a chemosensitizer protein for breast cancer cells in response to epidermal growth factor receptor‐tyrosine kinase inhibitor and suppresses tumor growth. The clinical information show nuclear factor I (NFI)‐C and NFI‐X expression correlate with AXL expression in breast cancer patients. Following, we establish serial deletions of AXL promoter to identify regions required for Adenovirus‐5 early region 1A (E1A)‐mediated AXL suppression. All of the NFI family members were extensively studied for their expression and functions in regulating AXL. Moreover, E1A post‐transcriptionally downregulates AXL expression through NFI. NFI‐C and NFI‐X, not NFI‐A and NFI‐B, resulting in cell death in response to EGFR‐TKI. Our finding suggests that NFI‐C and NFI‐X are crucial regulators for AXL and significantly correlated with poor survival of breast cancer patients.