Gamma-secretase represents a therapeutic target for the treatment of invasive glioma mediated by the p75 neurotrophin receptor

Abstract

The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target, suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.

Figure 1. p75^NTR Is Proteolytically Processed in Patient Specimens and Invasive Glioma Cells

(A) Western blot analysis detected p75^NTR-positive fragments migrating at 24 and 19 kDa in patient specimens expressing p75^NTR (eight of nine GBMs, two of five Grade III glioma) but was undetectable in tissue from normal human brain. U87 cells transfected with p75^NTR and grown in the presence of the proteasome inhibitor epoxomicin (1 μM) were used as a positive control (C). Western blots probed for beta-actin were used as a loading control. Molecular weight markers are indicated on the left. (B) The highly invasive glioma cell lines U87R and U251R isolated by serial in vivo selection were treated with the proteasome inhibitor epoxomicin (Epo, 1 μM) and/or the γ-secretase inhibitor, Compound X (CompX, 2 μM) for 4 h. Western blots for p75^NTR were probed with an antibody specific to the cytoplasmic domain of p75^NTR which detects full-length (75 kDa), CTF (indicated by the less than symbol [<]; 25 kDa), and ICD (indicated by the asterisk [*]; 19 kDa) peptides. The ICD is derived from the cleavage of the CTF, which is shown to visibly accumulate in the presence of a γ-secretase inhibitor. (C) p75^NTR proteolytic processing is a global event in human glioma cells. pcDNA3.1 encoding human p75^NTR or the empty pcDNA3.1 vector were stably transfected into U87, U251, U118, and U343. Cells expressing p75^NTR (U87p75, U251p75, U118p75, and U343p75) were treated as described above. Western blot analysis showed that all glioma cell lines tested cleaved the full-length p75^NTR to generate first the 24-kDa CTF and then the 19-kDa ICD.

Figure 2. γ-Secretase Inhibitor Significantly Inhibited p75^NTR-Induced Migration and Invasion in Glioma Cells

(A and B) Monolayer circular migration assays of U87R and U251R cells, which endogenously express p75^NTR, and U87p75 and U251p75 cells, which ectopically express p75^NTR, were performed. Briefly, cells were seeded in monolayer wells through a cell sedimentation manifold. p75^NTR-negative U87T, U87pcDNA, U251T, and U251pcDNA cells were used for comparison. Once the sedimentation manifolds were removed, cells were given complete medium containing 2 μM Compound X (CompX). Digital images of the cells were taken before migration at 0 h and then again at 72 h. Best-fit circles were drawn around the area covered by the cells at the 0 h and 72 h time points, and the actual cell area was determined using Axiovision 4.2 imaging software. Quantitative migration scores were calculated as the increase in the area covered by the cells beyond the initial area of the cells. γ-Secretase inhibitor Compound X significantly inhibited p75^NTR-induced migration. Values shown are the mean ± s.e.m. from three independent experiments; triple asterisks (∗∗∗) indicate p < 0.001 as compared to control (one-way analysis of variance [ANOVA] with the Neuman-Keuls post-test). (C and D) 3D-collagen invasion assays of U87R, U87p75, U251R, and U251p75 cells were performed by mixing cells with 3D-collagen matrix (collagen, fibronectin, and laminin) and then seeding them into 8.0-μm pore size transwell chambers in the presence or absence of 2 μM Compound X for 6 h. Cells were fixed and stained, and invasive cells were counted. U87T, U87pcDNA, U251T, and U251pcDNA cells were used for comparison. Inhibition of γ-secretase by Compound X significantly inhibited p75^NTR-induced glioma invasion. Values shown are the mean ± s.e.m. from three independent experiments; triple asterisks (***) indicate p < 0.001 as compared to control (one-way ANOVA with the Neuman-Keuls post-test).

Figure 3. Expression and Proteolytic Processing of p75^NTR Chimeric Alleles

(A) p75^NTR ICD is sufficient to induce invasion of U87 glioma cells in vitro. 3D-collagen invasion assays of U87p75ICD cells were performed by mixing cells with 3D-collagen matrix (collagen, fibronectin, and laminin) and then seeding them into 8.0-μm pore size transwell chamber for 6 h. Cells were fixed and stained, and invasive cells were counted. U87pcDNA and U87p75 cells were used for comparison. Values shown are the mean ± s.e.m. from three independent experiments; triple asterisks (***) indicate p < 0.001 as compared to U87pcDNA (one-way ANOVA with the Neuman-Keuls post-test). (B) Western blot analysis detected a p75^NTR-positive fragment migrating at 19 KDa in U87 cells expressing the p75-ICD construct. U87p75^NTR glioma cells treated with the proteasome inhibitor epoxomicin (Epo, 2 μM) were used as a positive control (U87-p75+Epo; the less than symbol [<] indicates CTF; the asterisk [*] indicates ICD). (C) Schematic diagram of the p75^NTR constructs. Chimeric proteins were created by replacing either the transmembrane (TM) or the extracellular stalk (S) domain of p75^NTR with equivalent domains from the Fas receptor. The proteins were designated p75FasTM (FasTM) and p75FasS (FasS), respectively. Other p75^NTR constructs were generated, including the neurotrophin-binding mutant in which the ligand-binding site was mutated by inserting four amino acids after amino acid residue 130, designated p75CRD130 (CRD130), and the p75^NTR intracellular domain construct consisting of amino acids 238 to 399 (p75-ICD). (D) Western blot analysis using a polyclonal p75^NTR antibody to the intracellular domain of p75^NTR confirmed the proper expression and processing of all p75^NTR constructs (p75FL, p75FasTM, p75FasS, or p75CRD130) in both U87 (left panel) and U251 (right panel) human glioma cell lines (the less than symbol [<] indicates CTF; the asterisk [*] indicates ICD). To examine comparable levels of chimeric proteins, lysates were adjusted accordingly.

Figure 4. Cleavage-Resistant Chimeric p75^NTR Proteins Do Not Induce Migration and Invasion In Vitro or In Vivo

(A and B) The invasive ability of U87 (A) and U251 (B) stably transfected with p75^NTR or the p75^NTR cleavage-resistant chimeric constructs (p75FasS and p75FasTM) were assessed using 3D-invasion assays. Expression of p75^NTR significantly increased invasion in the genetically distinct glioma cell lines U87 and U251, whereas neither p75FasS nor p75FasTM stimulated glioma invasion as compared to the p75^NTR-negative control cells (pcDNA). Values shown are the mean ± s.e.m. from three independent experiments; (the less than symbol [<] indicates CTF; the asterisk [*] indicates ICD; triple asterisks [∗∗∗] indicate p < 0.001 vs. p75^NTR wild type, one-way ANOVA with Neuman-Keuls post-test). (C) U87 cells stably expressing p75^NTR or the cleavage-resistant p75FasS and p75FasTM were implanted into the brains of SCID mice and allowed to grow for 28 d. The mice were sacrificed, and frozen brain sections were stained with antibodies against human nuclei (top row, brown) and human p75^NTR (bottom row, brown). Sections were counterstained with toluidine blue (blue). Scale bars in (C) represent 100 μm. Implantation of U87 glioma cells stably transfected with the empty pcDNA vector or p75^NTR cleavage-resistant chimeras (p75FasTM and p75FasS), led to the formation of well-circumscribed tumors. In marked contrast, animals implanted with U87 glioma cells ectopically expressing p75^NTR (U87p75) developed tumors with highly infiltrative edges. Similar results were seen in three independent experiments with three animals in each group. (D) p75^NTR cleavage-resistant chimeras do not generate ICD in vivo. Brain cryosections from SCID mice implanted with U87 glioma cells expressing p75^NTR, p75FasTM, or p75FasS were lysed in 2× loading buffer. Proteins were resolved on 10% SDS-PAGE gels and probed with a p75 cytoplasmic-specific antibody. U87R, U251R, and U87p75, which express high levels of p75^NTR and exhibit increased invasive activity, process full-length p75^NTR to generate both CTF and ICD in vivo. In contrast, and consistent with the in vitro data, the p75^NTR ICD peptide was not detected in tumors from animals expressing p75FasS or p75FasTM. Cell lysates from U87 cells expressing p75^NTR and grown in the presence of epoxomicin (p75^NTR+Epo) or expressing pcDNA (pcDNA) were used as positive and negative controls, respectively.

Figure 5. Regulated Intramembrane Proteolysis Occurs in Brain Tumor-Derived Stem-Like Cells (BTICs) from Glioma Patient Specimens

(A and B) Primary cultures from human glioma patient tumor grown under brain tumor stem cell-promoting conditions (BTIC) express high levels of p75^NTR (red) in vitro as detected by immunocytochemistry (A) and western blot (B). U87 cells stably expressing pcDNA and p75^NTR were used as negative and positive controls, respectively. Cultures were counterstained with DAPI (blue) to visualize the cell nucleus. (B) p75^NTR expressed on the BTICs undergoes RIP. BTICs were grown in the absence and presence of 2 μM γ-secretase and/or 1 μM epoxomicin. Western blot analysis detected full-length p75^NTR, CTF, and ICD with the accumulation of the 24-kDa CTF in the presence of the γ-secretase inhibitor (CompX). (C) Brain tumor-initiating cells (BTIC) express p75^NTR in vivo. BTIC cells were implanted into the brains of SCID mice and allowed to establish for 4–8 wk. Animals were sacrificed, and frozen sections were stained with anti-p75^NTR (brown). Sections were counterstained with toluidine blue (blue). Tumors established from BTICs express high levels of p75^NTR and form highly infiltrative tumors. M1 and M2 are individual mice.

Figure 6. γ-Secretase Inhibitor Significantly Blocked p75^NTR Induced Glioma Migration and Invasion In Vivo

(A–C) u87p75^NTR (A), U251p75^NTR (B), or p75^NTR-positive BTICs established from a patient GBM specimen (C) were implanted intracerebrally into SCID mice (m1–m3 and m7–m9 represent individual mice). Three days (U87p75^NTR and U251p75^NTR) or 5 d (BTIC) later, mice were administered s.c. 10 mg/kg γ-secretase inhibitor DAPT (γ-SI) or vehicle (corn oil) alone, once/day for 2–3 wk (three to five mice/group). The mice were sacrificed, and frozen brain sections were stained with an antibody against human p75^NTR (brown). Sections were counterstained with toluidine blue to visualize the cell nucleus (blue). Control animals given vehicle alone (corn oil; middle row) developed tumors with highly infiltrative edges (top panels). In marked contrast, animals that received daily injections of the γ-secretase inhibitor DAPT (bottom panels) developed localized tumors with demarcated edges. Similar results were seen in two independent experiments with three to five animals in each group. (D) γ-Secretase inhibitor blocks p75^NTR processing in vivo. Brain cryosections from three individual SCID mice implanted with u87p75^NTR-, U251p75^NTR-, or p75^NTR-positive BTICs and administered s.c. 10 mg/kg γ-secretase inhibitor DAPT or vehicle (corn oil) alone were lysed in 2× loading buffer. Proteins were resolved on 10% SDS-PAGE gels and probed with a p75 cytoplasmic-specific antibody. In animals given vehicle alone, western blot analysis detected p75^NTR-positive fragments migrating at 75, 24, and 19 kDa, whereas in cryosections from animals given the γ-secretase inhibitor DAPT, only the full-length p75^NTR and 25-kDa CTF were detected. U87 cells expressing p75^NTR and grown in the presence of epoxomicin (control; C) were used as a positive control. (The less than symbol [<] indicates CTF; the asterisk [*] indicates ICD.) (E) γ-Secretase inhibitor increases survival of animals bearing U87p75^NTR xenografts. Kaplan-Meier survival curves of SCID mice harboring U87p75^NTR intracranial tumors given s.c. injections of 10 mg/kg γ-secretase inhibitor DAPT or vehicle (corn oil) alone, once/day beginning on day 3. Animals given the γ-secretase inhibitor (GSI) survived significantly longer than control animals (p < 0.0001).