The E3 Ubiquitin Ligase NEDD4-1 Mediates Temozolomide-Resistant Glioblastoma through PTEN Attenuation and Redox Imbalance in Nrf2-HO-1 Axis.

Abstract

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to chemotherapy, and tumor recurrence is common. Neuronal precursor cell-expressed developmentally downregulated 4-1 (NEDD4-1) is an E3 ligase that controls embryonic development and animal growth. NEDD4-1 regulates the tumor suppressor phosphatase and tensin homolog (PTEN), one of the major regulators of the PI3K/AKT/mTOR signaling axis, as well as the response to oxidative stress. Methods: The expression levels of NEDD4-1 in GBM tissues and different cell lines were determined by quantitative real-time polymerase chain reaction and immunohistochemistry. In vitro and in vivo assays were performed to explore the biological effects of NEDD4-1 on GBM cells. Temozolomide (TMZ)-resistant U87MG and U251 cell lines were specifically established to determine NEDD4-1 upregulation and its effects on the tumorigenicity of GBM cells. Subsequently, miRNA expression in TMZ-resistant cell lines was investigated to determine the dysregulated miRNA underlying the overexpression of NEDD4-1. Indole-3-carbinol (I3C) was used to inhibit NEDD4-1 activity, and its effect on chemoresistance to TMZ was verified. Results: NEDD4-1 was significantly overexpressed in the GBM and TMZ-resistant cells and clinical samples. NEDD4-1 was demonstrated to be a key oncoprotein associated with TMZ resistance, inducing oncogenicity and tumorigenesis of TMZ-resistant GBM cells compared with TMZ-responsive cells. Mechanistically, TMZ-resistant cells exhibited dysregulated expression of miR-3129-5p and miR-199b-3p, resulting in the induced NEDD4-1 mRNA-expression level. The upregulation of NEDD4-1 attenuated PTEN expression and promoted the AKT/NRF2/HO-1 oxidative stress signaling axis, which in turn conferred amplified defense against reactive oxygen species (ROS) and eventually higher resistance against TMZ treatment. The combination treatment of I3C, a known inhibitor of NEDD4-1, with TMZ resulted in a synergistic effect and re-sensitized TMZ-resistant tumor cells both in vitro and in vivo. Conclusions: These findings demonstrate the critical role of NEDD4-1 in regulating the redox imbalance in TMZ-resistant GBM cells via the degradation of PTEN and the upregulation of the AKT/NRF2/HO-1 signaling pathway. Targeting this regulatory axis may help eliminate TMZ-resistant glioblastoma.