NOX4 regulates TGFβ-induced proliferation and self-renewal in glioblastoma stem cells

Glioblastoma (GBM) is the most aggressive and common glioma subtype, with a median survival of 15 months after diagnosis. Current treatments have limited therapeutic efficacy; thus, more effective approaches are needed. The glioblastoma tumoural mass is characterised by a small cellular subpopulation - glioblastoma stem cells (GSCs) - that has been held responsible for glioblastoma initiation, cell invasion, proliferation, relapse and resistance to chemo- and radiotherapy. Targeted therapies against GSCs are crucial, as is understanding the molecular mechanisms that govern the GSCs. Transforming growth factor β (TGFβ) signalling and reactive oxygen species (ROS) production are known to govern and regulate cancer stem cell biology. Among the differentially expressed genes regulated by TGFβ in a transcriptomic analysis of two different patient-derived GSCs, we found NADPH oxidase 4 (NOX4) as one of the top upregulated genes. Interestingly, when patient tissues were analysed, NOX4 expression was found to be higher in GSCs versus differentiated cells. A functional analysis of the role of NOX4 downstream of TGFβ in several patient-derived GSCs showed that TGFβ does indeed induce NOX4 expression and increases ROS production in a NOX4-dependent manner. NOX4 downstream of TGFβ regulates GSC proliferation, and NOX4 expression is necessary for TGFβ-induced expression of stem cell markers and of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), which in turn controls the cell's antioxidant and metabolic responses. Interestingly, overexpression of NOX4 recapitulates the effects induced by TGFβ in GSCs: enhanced proliferation, stemness and NRF2 expression. In conclusion, this work functionally establishes NOX4 as a key mediator of GSC biology.