Astrocyte-Specific Inactivation of the Neurofibromatosis 1 Gene (NF1) Is Insufficient for Astrocytoma Formation

Abstract

Individuals with the neurofibromatosis 1 (NF1) inherited tumor syndrome develop low-grade gliomas (astrocytomas) at an increased frequency, suggesting that the NF1 gene is a critical growth regulator for astrocytes. In an effort to determine the contribution of the NF1 gene product, neurofibromin, to astrocyte growth regulation and NF1-associated astrocytoma formation, we generated astrocyte-specific Nf1 conditional knockout mice (Nf1 GFAP CKO) by using Cre/LoxP technology. Transgenic mice were developed in which Cre recombinase was specifically expressed in astrocytes by embryonic day 14.5. Successive intercrossing with mice bearing a conditional Nf1 allele (Nf1flox) resulted in GFAP-Cre Nf1flox/flox (Nf1 GFAP CKO) animals. No astrocytoma formation or neurological impairment was observed in Nf1 GFAP CKO mice after 20 months, but increased numbers of proliferating astrocytes were observed in several brain regions. To determine the consequence of Nf1 inactivation at different developmental times, the growth properties of embryonic day 12.5 and postnatal day 2 Nf1 null astrocytes were analyzed. Nf1 null astrocytes exhibited increased proliferation but lacked tumorigenic properties in vitro and did not form tumors when injected into immunocompromised mouse brains in vivo. Collectively, our results suggest that loss of neurofibromin is not sufficient for astrocytoma formation in mice and that other genetic or environmental factors might influence NF1-associated glioma tumorigenesis.Neurofibromatosis 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with an incidence of 1 in 3,500 births worldwide (16). Early in life, individuals with NF1 develop café-au-lait spots, skinfold freckling, and iris hamartomas (Lisch nodules). In addition, approximately 15 to 20% of children with NF1 develop glial cell tumors (astrocytomas) involving the optic nerve, chiasm, hypothalamus, and brain stem (31). Although classified as grade I juvenile pilocytic astrocytomas, these tumors can be associated with significant morbidity as a result of visual loss or neurological compromise.Since individuals affected with NF1 develop tumors at an increased frequency, the NF1 gene is hypothesized to function as a tumor suppressor. Identification of the NF1 gene (10,42,43) and its protein product, the 220-to 250-kDa cytoplasmic neurofibromin protein, revealed that a small portion of the molecule has sequence similarity with the catalytic domain of a family of proteins termed GTPase-activating proteins (GAPs) (4,34,45). GAP molecules function as negative regulators of small mitogenic GTPase proteins, like p21 ras , and inactivate these signaling proteins by accelerating their conversion from active, GTP-bound conformations to inactive, GDP-bound forms (6). In NF1-associated tumors, loss of neurofibromin results in increased p21 ras mitogenic signaling and augments cell proliferation, leading to tumor formation (5,7,12).

The tumor suppressor neurofibromin confers sensitivity to apoptosis by Ras-dependent and Ras-independent pathways

“…[2][3][4][5] These abnormalities contribute to cell transformation in NF1 patients. 3,4 Studies in Nf1-deficient mice, [6][7][8] and Drosophila flies 9 provide additional support for the significance of aberrant Ras-GTP in NF1. Notably, the GAPrelated domain of neurofibromin (NF1-GRD) may not be the only neurofibromin domain underlying disease manifestations; for example, restoration of Ras signaling in Nf1 null mutant Drosophila flies does not correct the defect in body size, 10 and expression of neurofibromin in melanoma and NIH 3T3 cells inhibits their growth without affecting Ras-GTP levels.…”

The ecology of brain tumors: lessons learned from neurofibromatosis-1

“…In genetic pre-disposition models, GEM provides unprecedented opportunities to define the developmental changes in critical cell types throughout the natural history of tumor initiation, proliferation and progression. In this regard, mice engineered to lack Nf1 gene expression in glial fibrillary acidic protein-positive glial progenitor cells did not develop brain tumors (astrocytomas or gliomas) in vivo, although there was increased growth in Nf1-deficient astrocytes (Bajenaru et al, 2002). To more accurately model the NF1 human condition, mice heterozygous for an inactivating Nf1 mutation (Nf1 þ /À ) in every cell in their bodies were designed to also lack Nf1 gene expression (Nf1 À/À ) in glial progenitors (Bajenaru et al, 2003;Zhu et al, 2005).…”

Somatic neurofibromatosis type 1 (NF1) inactivation events in cutaneous neurofibromas of a single NF1 patient

“…5 However, no gliomas are formed in mice when the Nf1 gene is inactivated in astrocytes in the absence of a heterozygous background, indicating the need for cooperation of the Nf1+/ − non-neoplastic cells in the tumor microenvironment. 6 In contrast, investigation of humans with mosaic NF1 mutations shows that neurofibromas and Café-au-lait macules appear also within a wild-type background. 7 In addition, other factors such as the time point of biallelic inactivation or the expression of modifier genes seem to influence the formation of neurofibromas.…”