The p75 neurotrophin receptor is a central regulator of glioma invasion

Abstract

The invasive nature of cancers in general, and malignant gliomas in particular, is a major clinical problem rendering tumors incurable by conventional therapies. Using a novel invasive glioma mouse model established by serial in vivo selection, we identified the p75 neurotrophin receptor (p75(NTR)) as a critical regulator of glioma invasion. Through a series of functional, biochemical, and clinical studies, we found that p75(NTR) dramatically enhanced migration and invasion of genetically distinct glioma and frequently exhibited robust expression in highly invasive glioblastoma patient specimens. Moreover, we found that p75(NTR)-mediated invasion was neurotrophin dependent, resulting in the activation of downstream pathways and producing striking cytoskeletal changes of the invading cells. These results provide the first evidence for p75(NTR) as a major contributor to the highly invasive nature of malignant gliomas and identify a novel therapeutic target.

Figure 1. Serial In Vivo Selection Was Used to Isolate a Highly Invasive Glioma Population from a Noninvasive Human Malignant Glioma Cell Line

(A) The noninvasive human glioma cell line U87 stably expressing GFP (U87GFP) was implanted into the brains of SCID mice. Four to 6 wk later, the mice were sacrificed. The ipsilateral side of the brain (containing a grossly visible tumor) was separated from the contralateral side (containing only isolated invasive glioma cells [i.e., no macroscopically visible GFP-labeled tumor]), and both were grown in culture. These noninvasive (tumor) and highly invasive glioma cells were reimplanted into additional mice, and the process was repeated to select for increasingly noninvasive or invasive glioma cells. RNA extracted from the resulting invasive and tumor populations was used to prepare labeled cDNA that was hybridized to oligonucleotide microarrays. (B) Brains of SCID mice implanted with either tumor (left) or invasive (center and right) glioma cells. GFP visualization reveal the well-circumscribed border of the reimplanted tumor cells, with no tumor cells being detected away from the main tumor mass (left). This is in sharp contrast to the highly invasive border of the invasive tumors, where isolated small groups of glioma cells are found throughout the brain (center and right). Scale bars on GFP images represent 125 μm (center) and 62 μm (right and left).

Figure 2. Microarray Experiments Were Performed to Compare the Gene Expression Differences between the In Vivo–Selected Noninvasive (Tumor) and Invasive Glioma Cells

(A) Table lists results from a representative set of lineage experiments. Four independent microarray experiments were performed, each containing a pair of dye-flipped hybridizations. Genes that displayed consistent gene expression changes (>2-fold change in at least five out of eight hybridizations) are listed. The indicated ratios represent the fold change in gene expression in the invasive compared to the noninvasive cells. Genes chosen for validation are indicated in red. (B and C) Seven genes were chosen for validation; the expression of five are shown. (B) RT-PCR confirmed the expression of granulocyte colony-stimulating factor (G-CSF), interleukin-8 (IL-8), an unknown hypothetical protein DZFKp434B204 (DZFK), and tissue inhibitor of metalloproteinases-3 (TIMP-3) in the invasive population. Expression levels of GAPDH (unchanged) are shown for comparison. (C) RT-PCR and Western blot confirm expression of p75 in the invasive population (Inv) but not the tumor cell population (T). RT-PCR analysis of GAPDH levels and Western blot analysis of pyruvate kinase levels are included as loading controls. Human dorsal root ganglia (DRG) were used as a positive control. (D) Addition of NGF (200 ng/ml) enhanced the migratory ability of the invasive glioma cells in matrigel-coated invasion chambers, but had no significant effect on invasion of the tumor cells. Values shown are the mean ± SEM from three independent experiments. Triple asterisks (***) indicate p < 0.001 versus control (two-way ANOVA with Bonferroni post-tests).

Figure 3. p75^NTR Induces Migration and Invasion In Vitro

(A) Down-regulation of p75^NTR using RNAi decreases glioma migration/invasion. RT-PCR (GAPDH used as a loading control) and Western blot (pyruvate kinase used as a loading control) for p75^NTR confirm down-regulation of p75^NTR in the glioma cell line SF767 transfected with a p75-specific siRNA. Untransfected cells, cells transfected with a random siRNA, and the in vivo–selected tumor and invasive cells are shown for comparison. (B) Treatment with NGF (200 ng/ml) enhanced migration of SF767 cells transfected with the random (control) siRNA, but had no significant effect on migration of SF767 cells in which p75^NTR expression was inhibited by p75^NTR-specific siRNA. Values shown are the mean ± SEM from three independent experiments. A single asterisk (*) indicates p < 0.05, and double asterisks (**) indicate p < 0.01 versus control-treated random siRNA-transfected cells; triple asterisks (***) indicate p < 0.001 versus NGF-treated random siRNA-transfected cells (two-way ANOVA with Bonferroni post-tests). (C) Treatment with NGF (200 ng/ml) enhanced invasion of SF767 cells transfected with random siRNA, but had no significant effect on invasion of SF767 cells in which p75^NTR expression was inhibited by p75^NTR-specific siRNA. Values shown are the mean ± SEM from a single experiment. Similar results were seen in two independent experiments. Double asterisks (**) indicate p < 0.01 versus control-treated random siRNA-transfected cells, and double pluses (++) indicate p < 0.01 versus NGF-treated random siRNA-transfected cells (two-way ANOVA with Bonferroni post-tests). (D) Ectopic expression of p75^NTR induces glioma migration/invasion. RT-PCR (GAPDH used as a loading control) and Western blot (pyruvate kinase used as a loading control) for p75^NTR confirm expression of p75^NTR in U87 cells stably transfected with pcDNA3.1 encoding human p75^NTR (U87p75). Cells stably transfected with the empty pcDNA3.1 vector (U87pcDNA), as well as in vivo–selected tumor and invasive cells are shown for comparison. (E) Migration of U87 glioma cells is enhanced by ectopic expression of p75^NTR. No additional increase was seen following treatment with NGF (200 ng/ml). Values shown are the mean ± SEM from three independent experiments. Triple asterisks (***) indicate p < 0.001 versus pcDNA-transfected cells (two-way ANOVA with Bonferroni post-tests). (F) Similarly, invasion of U87p75 glioma cells in matrigel-coated invasion chambers was significantly increased compared to controls. No further increase was seen with exogenous NGF (200 ng/ml). Values shown are the mean ± SEM from four independent experiments. A single asterisk (*) indicates p < 0.05, and double asterisks (**) indicate p < 0.01 versus pcDNA-transfected cells (two-way ANOVA with Bonferroni post-tests).

Figure 4. Expression of p75^NTR in the U87 and U251N Glioma Cell Lines Dramatically Increases Invasion In Vivo

U87 or U251N human glioma cells stably transfected with the empty pcDNA vector (U87pcDNA [A] or U251NpcDNA [C]) or the p75^NTR-expression vector (U87p75 [B] or U251NpcDNA [D]) 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 (left) and human p75^NTR (right). Boxed areas indicate the region shown in the panel below, thus magnification increases from top to bottom; scale bars in (A) and (B) represent 100 μm, 50 μm, and 25 μm; and scale bars in (C) and (D) represent 200 μm and 100 μm. Implantation of U87 glioma cells stably transfected with the empty pcDNA vector led to the formation of well-circumscribed tumors that were p75^NTR negative (A). In sharp contrast, implantation of U87 glioma cells ectopically expressing p75^NTR led to the formation of tumors with highly infiltrative edges (B). Similar results were seen in three independent experiments with six animals in each group. U251NpcDNA glioma cells were generally more invasive than U87pcDNA cells upon implantation into the brains of SCID mice, and formed tumors with finger-like projections extending into the surrounding normal brain (C); nevertheless, ectopic expression of p75^NTR in U251N cells dramatically increased the invasiveness of these cells in vivo with isolated individual tumor cells being found distant from the main tumor mass (D). Similar results were seen in all ten animals in each group.

Figure 5. p75^NTR-Induced Glioma Invasion Is Neurotrophin Dependent

(A) U87 human glioma cells stably transfected with wild-type p75^NTR (red), the neurotrophin binding mutant p75CRD¹⁰⁵ (green), and p75CRD¹³⁰ (blue) or pcDNA empty vector (yellow) were examined for cell surface expression of p75 receptors by flow cytometry using the p75-specific primary antibody and an Alexa 448–conjugated secondary antibody. (B) Expression of BDNF in the conditioned medium (blue bar) and the total cell lysate (yellow bar) of U87 glioma cells expressing pcDNA (control), p75^NTR (p75), CRD 105, and CRD 130 were analyzed by ELISA. BDNF was found in the cell-associated fraction of U87 cells expressing p75, but not in the cells expressing the neurotrophin binding mutants CRD¹⁰⁵ or CRD¹³⁰. (C) U87 cells expressing wild-type p75^NTR, neurotrophin binding mutant CRD¹⁰⁵, or CRD¹³⁰ 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 (ANA; top row) and human p75^NTR (bottom row). Scale bars in (C) represent 100 μm. Implantation of either U87 glioma cells stably transfected with CRD¹³⁰ or CRD¹⁰⁵ vector, in contrast to the highly infiltrative edges of U87p75, led to the formation of well-circumscribed tumors. Similar results were seen in three independent experiments with six animals in each group. Expression of neurotrophin binding mutants of p75^NTR negates p75-induced glioma invasion.

Figure 6. p75^NTR Is Present and Confers Increased Migratory Ability in Glioblastoma Multiforme Patient Specimens

(A) Expression of p75^NTR (brown) was examined by immunohistochemistry in normal human brain specimens (zero of five) and GBM patient specimens (17 of 20). Representative samples are shown. Slides were counterstained with hematoxylin (blue). Scale bars from top to bottom represent 100 μm, 50 μm, and 25 μm. (B) p75^NTR mRNA and protein were assessed in glioblastoma patient specimens (GBM) and normal human brain (N). Human dorsal root ganglia (DRG) were used as a positive control. GAPDH was used as an internal loading control for RT-PCR. β-Tubulin was used as a protein loading control. Neuronal (neurofilament 70: [NF70]) and glial (glial fibrillary acid protein [GFAP]) markers in patient specimens are shown. (C) p75^NTR-positive glioma cells from patient specimens have an increased migratory ability. Migration of in vitro–cultured glioma patient specimens was evaluated using transwell motility assays. As a positive control for the assay, a mixture of 25% U87p75 cells and 75% U87pcDNA cells were analyzed at the same time as the patient samples. For both the control sample and the patient samples, the percentage of p75^NTR-positive cells in the migratory population (cells that migrated to the underside of the transwell) was increased compared to the percentage of p75^NTR-positive cells in the original population. Values shown are the mean ± SEM. A single asterisk (*) indicates p < 0.05; double asterisks (**) indicate p < 0.01; and triple asterisks (***) indicate p < 0.001 (t-test within a given sample).

Figure 7. Ectopic Expression of p75 Results in Actin Cytoskeletal Rearrangement and Decreased RhoA Activity

(A) Actin staining of tumor (left) and invasive cells (right) shows striking cytoskeletal rearrangement in the invading glioma cells. Actin cytoskeleton was visualized by staining fixed and permeabilized cells with rhodamine phalloidin (red), and cell nuclei were visualized with DAPI (blue). Numerous filamentous protrusions are seen in the invading glioma cells. (B) p75^NTR is sufficient to induce cytoskeletal rearrangement of glioma cells. U87pcDNA (left) and U87p75 (right) cells were fixed, permeabilized, and stained with rhodamine phalloidin (red) and DAPI to visualize the nucleus (blue). (C) p75^NTR expression results in decreased RhoA activity. RhoA activity was determined in U87pcDNA (pcDNA) and U87p75 (p75^NTR) by RhoA pulldown assay using a GST fusion protein containing RBD-rhotekin that binds only to activated (GTP-bound) RhoA. Western blots using total cell lysates were performed for p75^NTR, RhoA, and β-actin (used as a protein loading control). (D) Bar graph shows quantitation of activated RhoA (Rho-GTP) as compared to total RhoA in both U87pcDNA and U87p75. Values shown are the mean ± SEM from four independent experiments.