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. 2016 Mar 1;14(8):1979-90.
doi: 10.1016/j.celrep.2016.01.074. Epub 2016 Feb 18.

Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation

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Free PMC article

Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation

Jianqiang Wu et al. Cell Rep. .
Free PMC article

Abstract

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Genes identified by Sleeping Beauty Transposon System predict STAT3 and WNT pathway activation in neurofibroma
(A) Common insertion sites (CIS) identified from neurofibromas. The positions were based on the Ensembl NCBI m37 April 2007 mouse assembly. (B) Genemania pathway analysis using 22 CIS (black) and 20 genes (grey) connected to CIS by genetic, physical or pathway analysis identifies two significantly deregulated networks. WNT signaling related gene names are shown in Red and Cellular carbohydrate metabolic process related genes are in pink. GSK3b (turquoise) connects these pathways to those in Supplemental Figure 2. Circle size correlates to network association probabilities.
Figure 2
Figure 2. P-STAT3 and β-catenin expression correlate in mouse and human neurofibromas
(A) P-Stat3 immunostaining in mouse neurofibromas and wild type sciatic nerves (insert), visualized with DAB (brown). Nuclei are counterstained with hematoxylin (blue). (B) Representative immunofluorescent images show EGFP+ SCs (white arrows); some are also P- Stat3+ (purple) and β-catenin+ (red). Neurofibromas from 3 mice (4 sections/tumor) were stained. 350–500 EGFP+ cells were counted per section. DAPI (blue) staining highlights nuclei. Mean ± SEM is shown. (C) Western blot of P-Stat3-Y705 (P-Stat3) and total Stat3 (Stat3) in mouse neurofibroma (tumor) and wild-type sciatic nerve (WT nerve); blot is representative of neurofibromas (n=5) and wild-type nerves (n=3). (D) Representative pictures of immunostaining of P-STAT3(Y705) (left) and β-catenin (right) in human plexiform neurofibroma. In right, black arrow indicates nuclear β-catenin, white arrow indicates cytoplasmic β-catenin, black arrowhead indicates both. (E) Distribution of % P-STAT3 positive neurofibroma cells vs % nuclear β-catenin positive only (blue) or cytoplasmic and/or nuclear β-catenin positive (red) in 30 human plexiform neurofibromas. Spearman correlation coefficient analysis between P-STAT3(Y705) and β-catenin distribution (P-STAT3 vs total beta catenin shown in red, r=0.7219, p<0.0001; P-STAT3 vs nuclear beta catenin shown in blue, r=6374, p=0.0002, two tailed). (F) Quantification of intensity of P-STAT3(Y705) and β-catenin immune-positive cells in NF1 human plexiform neurofibromas and Spearman correlation coefficient analysis between P-STAT3(Y705) and β-catenin intensity (r=0.4373; p=0.0157, two-tailed). Bar=10μm.
Figure 3
Figure 3. Targeted genetic deletion of Stat3 in SCs and SCPs delays neurofibroma formation in vivo
(A) Kaplan-Meier survival curve. Purple: Stat3 fl/fl;Nf1fll+;DhhCre; Red, Stat3fl/fl;Nf1fl/fl;DhhCre; Blue: Stat3 fl/+;Nf1fl/fl;DhhCre. Green Nf1fl/fl;DhhCre. (B) Representative gross dissections of thoracic paraspinal neurofibromas and nerve roots in 9 month old Nf1fl/fl;DhhCre (left) and Stat3fl/fl;Nf1fl/fl;DhhCre (right) mice. Ruler shows 1 mm markings. (C) Neurofibroma volumes at 4, 7, and 9 months of age, measured in MRI images. Nf1fl/fl;DhhCre mice (n=12, black bars) and Stat3fl/fl;Nf1fl/fl;DhhCre mice (n=15, white bars) at 4, 7 and 9 months of age. (D) Average tumor number per mouse at 5 months in the Stat3fl/fl;Nf1fl/fl;DhhCre (white bar, n=6) and littermates Stat3fll+;Nf1fl/fl;DhhCre mice (black bar, n=6). (E) Tumor diameter in the Stat3fl/fl;Nf1fl/fl;DhhCre (circle, n=6 mice with 57 tumors) and littermates Stat3fll+;Nf1fl/fl;DhhCre mice (triangle, n=6 mice with 21 tumors). (F) Cell proliferation shown as percent Ki67+ cells in Nf1fl/fl;DhhCre mice (n=4, black bar) and Stat3fl/fl;Nf1fl/fl;DhhCre mice (n=3, white bar). (G) Cell death shown as percent cleaved caspase 3+ cells in Nf1fl/fl;DhhCre mice (n=4, black bar) and Stat3fl/fl;Nf1fl/fl;DhhCre mice (n=3, white bar). (H) Western blot on FACS-sorted EGFP+, EGFP- cells dissociated from adult Stat3fl/fl;DhhCre;EGFPfl/fl mouse sciatic nerves. Statistics: c: Ordinary one way ANOVA, d-g: unpaired student t test. *=p<0.05, **=p<0.01, **=p<0.001, n.s.=not significant.
Figure 4
Figure 4. Stat3 contributes to neurofibroma initiation
(A) The JAK2/STAT3 inhibitor FLLL32 inhibits formation of human neurofibroma spheres. DMSO (0) served as control. (B) Phase contrast images of human neurofibroma spheres treated with FLLL32 for 5 days. (C) Western blot of P-STAT3-Y705 and STAT3 in human neurofibroma spheres, +/−0.3 μM FLLL32. (D) Low doses of FLLL32 inhibit formation of mouse E12.5 Nf1−/− spheres; effects on E12.5 wild-type spheres are observed only at higher concentrations. (E) Phase contrast images of primary neurofibroma/DRG spheres from Stat3fl/+;Nf1fl/fl;DhhCre mice (left; control) and Stat3fl/fl;Nf1fl/fl;DhhCre mice (right). (F) Primary and secondary neurofibroma/DRG sphere number is reduced in the absence of Stat3 (n=3/group). (G) Neurofibroma-like lesions form after subcutaneous injection of Nf1fl/fl;DhhCre neurofibroma sphere cells but not in Stat3fl/fl;Nf1fl/fl;DhhCre mouse derived neurofibroma sphere cells. (H) Gross photograph of a lesion (black arrow) under reflected skin in a mouse injected with Nf1fl/fl;DhhCre neurofibroma sphere cells. Ruler shows 1 mm markings. (I) H & E stained section of F; lesion is indicated by white dotted line. (J) Immunohistochemistry showing S100β+ cells (brown) in tumor. Blue is hematoxylin counterstain. (K) Toluidine blue staining showing mast cells (black arrow). On EM, lesions contain SCs, identified by continuous basal lamina and wrapping of small axons (L), blood vessels (M) and mast cells (N). Bar=50μm B and E, others bar=20μm. Statistics: Ordinary one way ANOVA.
Figure 5
Figure 5. β-catenin signaling is a critical indicator of Stat3 in neurofibroma
(A) Western blots of nuclear and cytoplasmic proteins from Stat3fl/fl;Nf1fl/fl;DhhCre or Nf1fl/fl;DhhCre mouse neurofibromas/DRGs with indicated antibodies. (B) β-catenin tankyrase inhibitor XAV-939 inhibits formation of Nf1fl/fl;DhhCre mouse neurofibroma spheres. DMSO (0) was control. Insert: 10nM XAV-939 inhibited β-catenin expression by 3 days. (C) Low dose XAV-939 has no effect on the formation of Stat3fl/fl;Nf1fl/fl;DhhCre mouse neurofibroma spheres. (D) Three shβ-catenin shRNAs (#88, #89, and #90) each decrease mouse neurofibroma sphere formation, versus non-target lentivirus YFP (NT) or no virus controls. Insert shows Western blot confirming shβ-catenin-mediated knock down of total β-catenin. Anti-β-actin is loading control. Numbers show the ratio of β-catenin to β-actin loading control, then to no virus expression level. (E) Overexpression of β-catenin deltaN90 (ΔN90) in Stat3fl/fl;Nf1fl/fl;DhhCre mouse neurofibroma/DRG spheres increased sphere numbers (black) versus virus (grey, p<0.001) or no virus controls (white, p<0.001). Overexpression of ΔN90 β-cat in Nf1fl/fl;DhhCre mouse neurofibroma/DRG spheres did not significantly increase sphere numbers (black) versus virus (grey, p=0.15) or no virus (white, p=0.43). Insert: Western blot detects endogenous 92KDa β-catenin and ~60KDa of overexpression of mutated ΔN90 β-cat in Stat3fl/fl;Nf1fl/fl;DhhCre mouse neurofibroma spheres. Statistics: Ordinary one way ANOVA. Three independent experiments were performed in B, C and E.
Figure 6
Figure 6. Stat3 transcriptionally represses Arid1b expression, activating β-catenin
(A) qRT-PCR shows high Arid1b mRNA in Stat3fl/fl;Nf1fl/fl;DhhCre mouse sciatic nerve (white, n=6) versus WT (n=6) and Nf1fl/fl;DhhCre nerve (n=6). (B) Schematic, exon 1 mouse Arid1b gene. A putative Stat3 binding motif is between Exon 1 and Exon 2; the binding motif sequence is shown in bold. (C) Stat3 on the Arid1b promoter. PCR amplified a 139-bp Arid1b DNA fragment after ChIP with anti-Stat3. IgG was negative control. (D) Two Arid1b shRNAs increase numbers of Stat3fl/fl;Nf1fl/fl;DhhCre mouse neurofibroma spheres. (E) Western blot shows knockdown of Arid1b and increased β-catenin in Stat3fl/fl;Nf1fl/fl;DhhCre mouse neurofibroma spheres 4 days after shArid1b infection in two different shRNA clones. (F–G) β-catenin target gene expression increases after shArid1b (F) or shGsk3β (G) infection of Stat3fl/fl;Nf1fl/fl;DhhCre neurofibroma spheres. Mean ± SEM is shown for 3 independent experiments and 2 clones of shRNA in D, F and G. A representative experiment (of 3) is shown in C. Statistics: Ordinary one way ANOVA.
Figure 7
Figure 7. Arid1b and Gsk3β contribute to Stat3 mediated neurofibromagenesis
(A) In vitro, shGsk3β does not fully rescue Stat3fl/fl;Nf1fl/fl;DhhCre sphere numbers. Simultaneous knockdown of Gsk3β and Arid1b shows similar effects to shArid1b alone. (B) Neurofibroma-like tumors form in Stat3fl/fl;Nf1fl/fl;DhhCre sphere cells infected with shArid1b, shGsk3β, or both and transplanted into nu/nu mice. (C) Brg1 is necessary for Stat3fl/fl;Nf1fl/fl;DhhCre sphere formation in cells treated with shArid1b. (D) CHIP shows enhancement of the H3K4Me3 mark at the Arid1b promoter. (F) Schematic shows a model of neurofibroma initiation: Loss of Nf1 in SCP causes activation of P-Stat3. P-Stat3 transcriptionally represses Arid1b and Gsk3β, increasing β-catenin activity. Mean ± SEM is shown for 3 independent experiments in Panels A, C, and D. Two different shRNA clones (#1 and #2) were used in A and C, For combination, we used shArid1b #1+shGsk3β #1 (white bar) or shArid1b #2+shGsk3β #2 (grey bar) in A. We used shArid1b #1+shBrg1 #1 (white bar) shArid1b #2+shBrg1 #2 (grey bar) on C. Statistics: Ordinary one way ANOVA.

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