Transgenic mice overexpressing neuregulin-1 model neurofibroma-malignant peripheral nerve sheath tumor progression and implicate specific chromosomal copy number variations in tumorigenesis

Abstract

Patients with neurofibromatosis type 1 (NF1) develop benign plexiform neurofibromas that frequently progress to become malignant peripheral nerve sheath tumors (MPNSTs). A genetically engineered mouse model that accurately models plexiform neurofibroma-MPNST progression in humans would facilitate identification of somatic mutations driving this process. We previously reported that transgenic mice overexpressing the growth factor neuregulin-1 in Schwann cells (P(0)-GGFβ3 mice) develop MPNSTs. To determine whether P(0)-GGFβ3 mice accurately model human neurofibroma-MPNST progression, cohorts of these animals were monitored through death and were necropsied; 94% developed multiple neurofibromas, with 70% carrying smaller numbers of MPNSTs. Nascent MPNSTs were identified within neurofibromas, suggesting that these sarcomas arise from neurofibromas. Although neurofibromin expression was maintained, P(0)-GGFβ3 MPNSTs exhibited Ras hyperactivation, as in human NF1-associated MPNSTs. P(0)-GGFβ3 MPNSTs also exhibited abnormalities in the p16(INK4A)-cyclin D/CDK4-Rb and p19(ARF)-Mdm-p53 pathways, analogous to their human counterparts. Array comparative genomic hybridization (CGH) demonstrated reproducible chromosomal alterations in P(0)-GGFβ3 MPNST cells (including universal chromosome 11 gains) and focal gains and losses affecting 39 neoplasia-associated genes (including Pten, Tpd52, Myc, Gli1, Xiap, and Bbc3/PUMA). Array comparative genomic hybridization also identified recurrent focal copy number variations affecting genes not previously linked to neurofibroma or MPNST pathogenesis. We conclude that P(0)-GGFβ3 mice represent a robust model of neurofibroma-MPNST progression useful for identifying novel genes driving neurofibroma and MPNST pathogenesis.

Figure 1

P₀-GGFβ3 mice develop multiple neurofibromas that may undergo malignant transformation. A: Low-power image of a representative neurofibroma developing on a dorsal spinal nerve root in a P₀-GGFβ3 mouse. B: Higher-power view of the same tumor section, showing entrapped ganglionic neurons (arrows). C: In a section of the same tumor, immunostaining for S100β reveals coexistence of S100β-positive and -negative elements within this neoplasm. D: In a section of the same tumor, Unna’s methylene blue stain reveals numerous mast cells, evident by their prominent metachromasia (cells with dark purple granules) (arrows). E: In another tumor, immunostaining with the anti-neurofilament antibody SMI34 reveals axons (arrows) entrapped by infiltrating tumor cells. F: Microscopic focus of apparent malignant transformation, a markedly hypercellular region (left half of the field), arising in a spinal nerve root neurofibroma. Scale bars: 500 μm (A); 50 μm (B–F).

Figure 2

Representative images of MPNSTs arising in P₀-GGFβ3 mice, demonstrating the histological variability encountered in these neoplasms. A and B: Histology of tumors that arose independently in the left (A) and right (B) trigeminal nerves of mouse B76. Note the extreme spindled morphology of the tumor cells in A, which contrasts markedly with the densely packed, poorly differentiated cells composing the tumor seen in B. C: MPNST with an epithelioid morphology that arose in the sciatic nerve of mouse A202. D: A trigeminal MPNST from mouse A387 invading the undersurface of the brain. The overlying brain parenchyma (asterisk) is being invaded by this tumor. Scale bars: 50 μm (A–C); 200 μm (D).

Figure 3

Early-passage P₀-GGFβ3 MPNST cells consistently express multiple Schwann cell markers and variably express neural and muscle markers. A and B: Early-passage cultures of tumors arising on mixed C57BL/6×SJL (A390 cells) (A) or inbred C57BL/6 (B91 cells) (B) backgrounds uniformly express the Schwann cell marker S100β (Cy3 immunoreactivity; red-orange staining). These cells have been counterstained with the nuclear stain bisbenzimide (blue). C and D: Immunostaining of array sections reveals the expression of other markers, such as GFAP (C) and SMA (D), that was observed in some lines. Sections have been lightly counterstained with hematoxylin. E: RT-PCR for Schwann cell (S100β, P₀, MBP, PMP22, p75^LNTR, Sox10, Pax3, Krox20, GFAP, GAP43), neural (neurofilament, peripherin), and muscle (calponin, SM22α, α-SMA, desmin, MyoD1) markers in early-passage cultures established from 18 independently arising P₀-GGFβ3 MPNSTs. F-U: Double-label immunohistochemistry for S100β and neurofilaments (F–I), desmin (J–M), or SMA (N–Q) performed on the parent tumors (A391 and A387) used as the source of MPNST cultures that aberrantly expressed neuronal and/or muscle markers. For controls (R–U), immunostains were performed with secondary antibodies alone, in the absence of primary antibodies. Sections were counterstained with bisbenzimide (Bis).

Figure 4

Although neurofibromin expression is typically maintained in P₀-GGFβ3 MPNSTs, Ras is hyperactivated in these neoplasms, compared with WT Schwann cells. A: Immunoblotted lysates of WT and non-neoplastic transgenic Schwann cells and 18 early-passage P₀-GGFβ3 MPNST cultures probed for neurofibromin (1:500 antibody dilution). The 250-kDa form of neurofibromin is present in Schwann cells and the MPNST cells, with some MPNST cultures also exhibiting the 220-kDa splice variant of this protein. B: The Raf-1 Ras-binding domain (RBD) was used to pull down activated Ras protein in WT Schwann cells and three early-passage P₀-GGFβ3 MPNST cultures. An immunoblot of the captured activated Ras proteins was then probed with a pan-Ras antibody that detects H-Ras, K-Ras, and N-Ras (upper panel). The middle and lower panels show immunoblots of the clarified lysate before Ras pull-down probed with the pan-Ras antibody and an anti-GAPDH antibody, respectively.

Figure 5

Abnormalities in the p19^ARF–Mdm–p53 signaling pathway occur in early-passage P₀-GGFβ3 MPNST cultures. A–F: Immunocytochemistry reveals intranuclear accumulation of p53 in two representative p53-overexpressing P₀-GGFβ3 MPNST early-passage cultures. p53 immunoreactivity is indicated by red-orange staining (A and D); bisbenzimide was used as a nuclear counterstain (blue) (B and E). Merged images of p53 immunoreactivity and bisbenzimide staining (C and F) demonstrate that p53 in these tumor cells accumulates within the nuclei. G: Immunoblotted lysates of non-neoplastic transgenic Schwann cells and early-passage cultures derived from 18 independently arising P₀-GGFβ3 MPNSTs probed for p53 (1:1000 dilution). Blots were reprobed with GAPDH to verify equivalent loading. H: Immunoblotted lysates of non-neoplastic transgenic Schwann cells and early-passage cultures derived from 18 independently arising P₀-GGFβ3 MPNSTs probed for Mdm2 or Mdm4 (each at 1:500 dilution). Blots were reprobed with GAPDH to verify equivalent loading. Original magnification, ×40 (A–F).

Figure 6

Abnormalities in cell-cycle regulatory proteins are common in early-passage cultures of P₀-GGFβ3 MPNST cells, but these cells are persistently dependent on aberrant growth factor signaling. A: Immunoblotted lysates of non-neoplastic transgenic Schwann cells and early-passage cultures of P₀-GGFβ3 MPNST cells probed for cyclin D1 (1:300 dilution), cyclin D2 (1:500 dilution), cyclin D3 (1:700 dilution), CDK4 (1:500 dilution), CDK2 (1:400 dilution), p27^Kip1 (1:600 dilution), p21^Cip1 (1:500 dilution), or Rb phosphorylated on Ser⁷⁹⁵ (1:700 dilution). Blots were reprobed with GAPDH to verify equivalent loading. B: qPCR analyses of Cdkn2a expression in non-neoplastic transgenic Schwann cells and early-passage cultures of P₀-GGFβ3 MPNST cells. Transcript levels were normalized to levels detected in non-neoplastic transgenic Schwann cells. C: The ErbB inhibitor PD168393 inhibits DNA synthesis in early-passage cultures of P₀-GGFβ3 MPNST cells in a concentration-dependent fashion. [³H]thymidine incorporation relative to vehicle is shown for four early-passage cultures (A292, A231Tr, A231Sc, and A390) derived from MPNSTs arising in P₀-GGFβ3 mice. Data are expressed as means ± SEM. *P < 0.05 versus control. n.d., not detectable.

Figure 7

aCGH analysis of the 2.47-Mb region in chromosome 11 encoding the Trp53 tumor suppressor gene in P₀-GGFβ3 MPNST A18. The genomic map indicates the location of the genes present within the region illustrated in the accompanying panel, which indicates the relative signals in tumor (red) and non-neoplastic Schwann cell (green) DNAs in the MPNST. The height of the colored bar indicates the relative difference in these two signals. Note the central 1.21-Mb region in which there is a relative copy number loss in the tumor.

Figure 8

aCGH analyses of a 1.1-Mb region in chromosome 11 that surrounds a region of relative copy number loss present in five P₀-GGFβ3 MPNSTs (A202, A292, A382, B76, and B86). The genomic map indicates the location of the genes present within the region illustrated in the accompanying panels, which indicate the relative signals in tumor (red) and non-neoplastic Schwann cell (green) DNAs in the MPNSTs. The height of the colored bar indicates the relative difference in these two signals. Note the variably sized central regions in which there is a relative copy number loss in these five MPNSTs.

Figure 9

aCGH analysis of a 488-kb region in chromosome 11 that surrounds a region of relative copy number loss present in two P₀-GGFβ3 MPNSTs (A202 and B76). The genomic map indicates the location of the genes present within the region illustrated in the accompanying panels, which indicate the relative signals in tumor (red) and non-neoplastic Schwann cell (green) DNAs in the MPNSTs. The height of the colored bar indicates the relative difference in these two signals. Note the central 44-kb region in which there is a relative copy number loss in these two MPNSTs.

Figure 10

Ideogram indicating the locations of focal CNVs detected by aCGH data in early-passage cultures established from P₀-GGFβ3 MPNSTs. Regions of copy number gain are indicated in red, and regions of unbalanced loss are indicated in green. The lengths of the red and green bars are proportional to the size of the CNV.