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

Abstract

Background: The nuclear factor I-A (NFIA) transcription factor promotes glioma growth and inhibits apoptosis in glioblastoma (GBM) cells. Here we report that the NFIA pro-survival effect in GBM is mediated in part via a novel NFIA-nuclear factor-kappaB (NFκB) p65 feed-forward loop.

Methods: We examined effects of gain- and loss-of-function manipulations of NFIA and NFκB p65 on each other's transcription, cell growth, apoptosis and sensitivity to chemotherapy in patient-derived GBM cells and established GBM cell lines.

Results: NFIA enhanced apoptosis evasion by activating NFκB p65 and its downstream anti-apoptotic factors tumor necrosis factor receptor-associated factor 1 (TRAF1) and cellular inhibitor of apoptosis proteins (cIAPs). Induction of NFκB by NFIA was required to protect cells from apoptosis, and inhibition of NFκB effectively reversed the NFIA anti-apoptotic effect. Conversely, NFIA knockdown decreased expression of NFκB and anti-apoptotic genes TRAF1 and cIAPs, and increased baseline apoptosis. NFIA positively regulated NFκB transcription and NFκB protein level. Interestingly, NFκB also activated the NFIA promoter and increased NFIA level, and knockdown of NFIA was sufficient to attenuate the NFκB pro-survival effect, suggesting a reciprocal regulation between NFIA and NFκB in governing GBM cell survival. Supporting this, NFIA and NFκB expression levels were highly correlated in human GBM and patient-derived GBM cells.

Conclusions: These data define a previously unknown NFIA-NFκB feed-forward regulation that may contribute to GBM cell survival.

Keywords: NFκB; apoptosis; chemoresistance; glioblastoma (GBM); nuclear factor I-A (NFIA).

Fig. 1

NFIA enhances GBM cell survival. (A) Growth of GBM1 primary tumorspheres stably expressing NFIA or vector (also see Fig. S1). (B, C) GBM1 cells stably expressing NFIA or vector control (Cont) were treated with temozolomide (TMZ 25 μg/mL, B) or etoposide (ETP 1 μg/mL, C; Fig. S2) or vehicle (dimethyl sulfoxide [DMSO]) for 24 h. Apoptosis (%) was assessed using the Apo-Direct kit; means ± SD from 3 experiments. (D) NFIA inhibits caspase-mediated apoptosis induced by ETP; caspase-3 activity measured in GBM cells expressing NFIA or vector following treatment with ETP. (E) Caspase-8 cleavage measured in GBM cells expressing NFIA or vector following treatment with the increasing amount of etoposide (ETP; also see Fig. S3).

Fig. 2

NFIA increases NFκB p65 transcription and induces NFκB activity and nuclear translocation. (A) Putative NFIA binding sites in the human NFκB p65 promoter. (B) Relative mRNA expression of NFκB p65 to GAPDH in GBM cells expressing NFIA or vector was determined by RT-PCR and densitometric analysis. (C) Immunoblots of whole cell lysates from GBM1 cells transiently transfected with the increasing amount of a lentivector encoding NFIA (bottom; densitometry). (D) Luciferase activities were measured 48 h after transfection of NFκB p65 luciferase reporter into U87 GBM cells expressing NFIA, shNFIA, or controls (vector or shCont); n = 3. (E) Relative luciferase activity of pGL3 wild-type (wt) or mutant (mt) NFκB p65 promoter transfected into U87 GBM cells expressing shNFIA or shCont similar to the left panel. (F) ChIP-qPCR assays on GBM cells using anti-NFIA or control immunoglobulin G. (G) Luciferase assay with the 3x κB-luc-reporter in U87 cells transfected with NFIA constructs (NFIA, vector only, shNFIA, or shCont). (H) NFIA promotes nuclear translocation of NFκB p65. Immunofluorescence staining of GBM1 cells expressing HA-NFIA or vector control with Hoechst (nuclei; blue), anti-HA (green), anti-p65 NFκB (red): arrows: nuclear p65 in HA-positive cells, arrowheads: cytoplasmic p65 in HA-negative control cells.

Fig. 3

NFIA pro-survival effect is mediated through NFκB p65 (A) Caspase-3 activity measured in NFIA-deleted GBM cells (transduced with shNFIA or shCont) treated with GFP-NFκB p65 or GFP vector control. NFκB p65 and TRAF1 levels in GBM cells used in (A) were verified by immunoblotting (Fig. S4). (B, C) Immunoblots of GBM1, U251, and U87 GBM cells transduced with NFIA, vector control, shRNA (shNFIA), or shRNA control (shCont).

Fig. 4

NFκB p65 regulates NFIA transcription activity. (A) A putative NFκB p65-binding site in the human NFIA promoter luciferase reporter. (B) Luciferase activity was measured 48 h after transfection of NFIA luciferase reporter into U87 GBM cells expressing NFκB p65 siRNA (siNFκB p65) or control (siCont); n = 3; western blot of whole cell lysate of GBM cells used in the experiment B. (C) Relative luciferase activity of pGL3 wild-type (wt) or mutant (mt) NFIA promoter transfected into U87 cells expressing siNFκB p65 or siCont. (D) NFIA mRNA expression measured by RT-PCR in GBM cells transfected with NFκB p65 or vector control, n = 3. (E) ChIP-qPCR assays on GBM cells using anti-NFκB p65 or immunoglobulin G control.

Fig. 5

NFκB p65-induced inhibition of apoptosis is mediated in part by NFIA. (A, B). Apoptosis induced by knockdown (siNFκB) or pharmacologic inhibition (iNFκB) of NFκB is reversed by introduction of NFIA in U87 GBM cell (also see Fig. S5). Caspase-3 activity in NFκB p65-depleted GBM cells (siNFκB or siCont, or NFκB inhibitor or DMSO) transduced with NFIA or vector control (also see Fig. S6). (C) Immunoblots of whole cell lysates from GBM cells expressing GFP-p65 or GFP only. Right-densitometric analysis relative to GAPDH. (D) Knockdown of NFκB p65 decreases NFIA level. NFIA levels in GBM cells transfected with NFκB p65 siRNA (siNFκB p65) or siRNA control (siCont).

Fig. 6

Downregulation of NFIA sensitizes GBM cells to chemotherapy-induced cell death. (A) Knockdown of NFIA by shNFIA or NFκB p65 by siNFκB p65 decreased GBM tumorsphere formation compared with control (shCont or siCont). (B, C) Greater reduction in GBM tumorsphere formation in NFIA-depleted GBM cells treated with ETP or TMZ. GBM1 cells transduced with shNFIA or shCont, treated with TMZ or ETP for 24 h. (D) Downregulation of NFIA sensitized GBM cells to ETP-induced apoptosis. Apoptosis was measured using Apo-Direct kit followed by cell sorting analysis. (E) Greater reduction in GSC survival in NFIA-depleted GSCs treated with NFκB inhibitor (iNFκB). (F) Immunoblot of whole cell lysates from human GBM (n = 12) and nonneoplastic brains (n = 4). (G) Densitometric analysis of NFIA and NFκB p65 levels relative to GAPDH. (H) Immunoblot of whole cell lysates from patient derived GBM cells (GBM 1–5) and normal astrocytes (Fig. S8). (I) Correlation between NFIA and NFκB mRNA expression in GBM (580 GBM samples in total, 291 samples with completed information; n = 291; TCGA, Cell 2013; cbioportal.org). (J) Schematic model of an NFIA-NFκB feed-forward mechanism in GBM cell survival.