Spatiotemporal Loss of NF1 in Schwann Cell Lineage Leads to Different Types of Cutaneous Neurofibroma Susceptible to Modification by the Hippo Pathway

Abstract

Neurofibromatosis type 1 (NF1) is a cancer predisposition disorder that results from inactivation of the tumor suppressor neurofibromin, a negative regulator of RAS signaling. Patients with NF1 present with a wide range of clinical manifestations, and the tumor with highest prevalence is cutaneous neurofibroma (cNF). Most patients harboring cNF suffer greatly from the burden of those tumors, which have no effective medical treatment. Ironically, none of the numerous NF1 mouse models developed so far recapitulate cNF. Here, we discovered that HOXB7 serves as a lineage marker to trace the developmental origin of cNF neoplastic cells. Ablating Nf1 in the HOXB7 lineage faithfully recapitulates both human cutaneous and plexiform neurofibroma. In addition, we discovered that modulation of the Hippo pathway acts as a "modifier" for neurofibroma tumorigenesis. This mouse model opens the doors for deciphering the evolution of cNF to identify effective therapies, where none exist today. SIGNIFICANCE: This study provides insights into the developmental origin of cNF, the most common tumor in NF1, and generates the first mouse model that faithfully recapitulates both human cutaneous and plexiform neurofibroma. The study also demonstrates that the Hippo pathway can modify neurofibromagenesis, suggesting that dampening the Hippo pathway could be an attractive therapeutic target.This article is highlighted in the In This Issue feature, p. 1.

Figure 1.. Hoxb7 lineage-derived cell populate nerve ending in the dermis

(A-C) X-Gal staining was performed on E9.5 – E12.5 embryos (A) and adult mice (B-C) with genotype Hoxb7-Cre;LacZ. (A) Immunohistochemistry using anti-HOXB7 antibodies on transectional cut of E12.5 embryo at the chest (middle panels) and cranial (lower panels) level. (B) Gross dissection of adult mice peripheral nervous system. Black arrow indicates dorsal sensory nerves. White arrow heads indicates ventral roots. SC=Spinal Cord. (C) Immunohistochemistry using Schwann cell (S100β, GAP43) and neuronal (βIII tubulin) markers on histological section of dorsal root ganglion (DRG), intercostal nerve (IN) and skin X-Gal counterstained (blue). (D) Typical representation of human cNF (left panel). Scale bar in millimeters. Hematoxylin and eosin (H&E) and immunohistochemistry using anti-HOXB7 antibodies on human cNF (middle panels). Immunofluorescence on human cNF using anti-HOXB7 and anti-S100β antibodies. Scale bar = 50μm.

Figure 2.. Hoxb7 lineage derived Nf1−/− SKPs give rise to neurofibroma

(A) Diagram of experimental design: Isolation of SKPs from dorsal skin of newborns pups with Nf1^f/f;LacZ (left panel), Hoxb7-Cre;Nf1^f/f;LacZ (middle panel), Hoxb7-Cre;Nf1^f/f;YFP (right panel). Nf1^f/f;LacZ- SKPs and Nf1^−/−;LacZ+ or YFP+ SKPs were implanted on the left and right sciatic nerve of nude mice, respectively. H&E and immunohistochemistry to measure expression of Schwann cell marker (S100β, GAP43) and HOXB7 (HOXB7, GFP) were performed on histological sections of the right sciatic nerve. Scale bar = 50μm. (B) Estimation of enrichment of Hoxb7 lineage derived cells in neurofibroma. YFP+ and YFP- cells from SKPs harvested from Hoxb7-Cre;Nf1^f/f;YFP were analyzed by FACS to determine the basal level of YFP+ cells. YFP+ cells were evaluated in SKP-induced neurofibroma by immunohistochemistry.

Figure 3.. Ablation of Nf1 in Hoxb7 lineage cells give rise to diffuse cutaneous Neurofibroma

(A) H7;Nf1mut mouse model bearing diffuse cNF. (B) Dissection of H7;Nf1mut mice showing that tumors are contained within the dermis. (C) NF1 patient with diffuse cNF of the forearm. (D) Biopsy of human diffuse cNF showing that tumor is contain within the dermis. (E-F) Histological characterization of murine (E) and human (F) diffuse cNF. Histochemistry was performed with X-Gal staining (Hoxb7 lineage tracing in mice), H&E, Sirius red (collagen), toluidine blue (mast cells), and immunofluorescence with anti-S100β and anti-GAP43 (Schwann cell markers). Black and white scale bar = 50μm. Blue scale bar = 1mm.

Figure 4.. Ablation of Nf1 in Hoxb7 lineage cells give rise to plexiform Neurofibroma

(A) Dissected spinal cord and peripheral nerves from H7;Nf1mut. White arrow = DRG. Black arrow head = intercostal nerve. (B) X-Gal staining and immunohistochemistry with anti-S100β and anti-GAP43 (Schwann cell markers) on intercostal nerve (IN) (upper panels) and dorsal root ganglion (DRG) (lower panels) and from H7;Nf1mut. Black scale bar = 50μm. Blue scale bar = 1mm.

Figure 5.. Hippo pathway act as a modifier of cutaneous neurofibroma

(A-B) Hippo pathway mutation analysis of human cNF. 165 genes known to play a role in the Hippo pathway (literature search and KEGG) was used to mine genomic alterations identified in a dataset of 33 cutaneous neurofibroma collected from 9 individual NF1 patients. Hippo pathway mutations were plotted for both (A) germline (7 out of 9 patients have mutations) and (B) somatic datasets (7 out of 33 cNFs have additional somatic mutations). Colors indicate the type of mutation observed as described in the figure legend. (C) Immunohistochemistry of human discrete (upper panels) and diffuse (lower panels) cNF using anti-YAP and anti-TAZ antibodies. (D) Relative mRNA expression of AXL and CTGF by real-time PCR on 5 cNF and their adjacent normal margin. (E) Representative picture of H7;Nf1mut;Lats1/2mut mice that develop diffuse cNF (upper panel) and discrete cNF nodule (lower panel). Histological characterization of H7;Nf1mut;Lats1/2mut mice that develop diffuse cNF (upper panels) and discrete cNF nodule (lower panels) by H&E and immunofluorescence with anti-S100β and anti-GAP43 (Schwann cell markers). (F) Representative H&E used to measure the skin thickness in H7;Nf1mut (upper left panel) and H7;Nf1mut;Lats1/2mut (lower left panel). Scattered plot representing the skin thickness in function of genotype (right panel) ** = p < 0.01. (G) Bar graph representing the percentage of mice developing diffuse cNF in specific body location (left panel). Bar graph of the percentage of mice developing diffuse cNF in function of genotype * = p < 0.05 (right panel). Black and white scale bar = 50μm. Blue scale bar = 500μm. L1=Lats1. L2=Lats2.

Figure 6.. Hippo pathway act as a modifier of plexiform neurofibroma

(A, D) Picture of the cervical and trunk spinal cord and peripheral nerves (A) and sciatic nerves (D) of H7;Nf1mut (left panels), H7;Lats1/2mut (middle panels) and H7;Nf1mut;Lats1/2mut (right panels). (B, E) Histological evaluation of DRGs (B) and sciatic nerves (E) in function of the genotype. (C, F) Scattered plot of (C) the number of enlarged DRGs (> 1mm diameter) and (F) diameter of sciatic nerves as a function of genotype. (G) Kaplan-Maier plot illustrating the percentage of mice surviving as a function of time and genotype. ** = p < 0.01; *** = p < 0.001. Scale bar = 50μm.

Figure 7.. Hippo pathway inactivation enhances MAPK pathway activation induced by NF1 loss.

(A-B) Representative immunohistochemistry using anti p-ERK1/2 (upper panels) and anti-total ERK1/2 (lower panel) of (A) skin, (B) DRG of H7;Nf1mut (left panels), H7;Lats1/2mut (middle panels) and H7;Nf1mut;Lats1/2mut (right panels). Bar graphs (far right) indicate the quantification as percentage of positive cells found in A,B. (C) Western-Blot analysis using anti p-ERK1/2 and anti-total ERK1/2 in mouse embryonic DRG/Nerve root sphere cells from Lats1+/+;Lats2f/f and Lats1+/+;Lats2f/+ treated with adenovirus cre (Cre) or control adenovirus (GFP) (upper panel). Quantification as ratio of p-ERK1/2 over total ERK1/2 immunoblot (lower panel). (D) Western-Blot analysis using anti p-ERK1/2 and anti-total ERK1/2 in whole tissue extract from murine skin and DRG from wild type (WT), H7;Nf1mut, H7;Lats1/2mut and H7;Nf1mut;Lats1/2mut. Quantification as ratio of p-ERK1/2 over total ERK1/2 immunoblot (lower panel).