A novel tumor-promoting role for nuclear factor IA in glioblastomas is mediated through negative regulation of p53, p21, and PAI1

Abstract

Background Nuclear factor IA (NFIA), a transcription factor and essential regulator in embryonic glial development, is highly expressed in human glioblastoma (GBM) compared with normal brain, but its contribution to GBM and cancer pathogenesis is unknown. Here we demonstrate a novel role for NFIA in promoting growth and migration of GBM and establish the molecular mechanisms mediating these functions. Methods To determine the role of NFIA in glioma, we examined the effects of NFIA in growth, proliferation, apoptosis, and migration. We used gain-of-function (overexpression) and loss-of-function (shRNA knockdown) of NFIA in primary patient-derived GBM cells and established glioma cell lines in culture and in intracranial xenografts in mouse brains. Results Knockdown of native NFIA blocked tumor growth and induced cell death and apoptosis. Complementing this, NFIA overexpression accelerated growth, proliferation, and migration of GBM in cell culture and in mouse brains. These NFIA tumor-promoting effects were mediated via transcriptional repression of p53, p21, and plasminogen activator inhibitor 1 (PAI1) through specific NFIA-recognition sequences in their promoters. Importantly, the effects of NFIA on proliferation and apoptosis were independent of TP53 mutation status, a finding especially relevant for GBM, in which TP53 is frequently mutated. Conclusion NFIA is a modulator of GBM growth and migration, and functions by distinct regulation of critical oncogenic pathways that govern the malignant behavior of GBM.

A novel NFIA-NFκB feed-forward loop contributes to glioblastoma cell survival

“…U251MG and U87MG cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and L-glutamine (2 mM), and maintained in 5% CO 2 at 37°C. Human patient derived GBM cells (glioma sphere cultures [GSC]) were previously described 13,29 and were cultured in serum-free neurobasal media supplemented with N2, B27, and L-glutamine (Invitrogen), along with epidermal growth factor and basic fibroblast growth factor (20 ng/mL each). Cells were used according to the needs of the experimental design (ie, transduction efficacy, growth as monolayer, etc).…”

“…GBM tumorsphere assay was performed using human patient derived GSC expressing NFIA constructs (NFIA, vector, short hairpin NFIA [shN-FIA], and control shRNA [shCont]), and tumorspheres larger than 0.1 mm were quantified as described before. 13 Apoptosis Assays, Caspase-3 Activity Apoptosis was evaluated by labeling DNA breaks with fluorescein isothiocyanate-deoxyuridine triphosphate (FITC-dUTP) followed by flow cytometry, using the ApoDirect kit (BD Biosciences Pharmingen), according to the manufacturer's instructions. Caspase activity was measured using the ApoTarget Caspase-3 Colorimetric Protease Assay (BioSource), and was determined in 200 μg of lysate proteins according to the manufacturer's instructions.…”

“…[12][13][14] We and others reported that NFIA is highly expressed in astrocytomas of all grades compared with nonneoplastic brains and that high NFIA confers growth advantage in gliomas. We recently reported that NFIA is necessary and sufficient to promote glioma growth, proliferation, and inhibition of apoptosis in a manner mediated in part through negative regulation of tumor suppressors, such as p53 and p21, identifying NFIA as a critical component of the oncogenic network in glioma.…”

“…We recently reported that NFIA is necessary and sufficient to promote glioma growth, proliferation, and inhibition of apoptosis in a manner mediated in part through negative regulation of tumor suppressors, such as p53 and p21, identifying NFIA as a critical component of the oncogenic network in glioma. 13 The molecular mechanisms by which NFIA induces glioma cell survival and drug resistance are not fully understood.…”

“…13 To achieve efficient knockdown of endogenous NFIA, we used a lenti-shRNA specific to human NFIA (shNFIA) and compared it with a control shRNA (shCont) as previously described. 13 The human NFκB p65 (−507 to 67) and NFIA (−1400 to −450) promoter plasmids were generated using gene synthesis (Genewiz). The inserts were ligated into pGL3-basic vector (Promega) using KpnI/XhoI sites.…”