- Email: [email protected]
Neurofibromatosis 1: closing the GAP between mice and men
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Neuro®bromatosis 1: closing the GAP between mice and men Biplab Dasgupta and David H Gutmann Neuro®bromatosis 1 (NF1) is a common genetic condition in which affected individuals are prone to the development of benign and malignant tumors. The NF1 tumor suppressor encodes a protein product, neuro®bromin, which functions in part as a negative regulator of RAS. Loss of neuro®bromin expression in NF1-associated tumors or Nf1-de®cient mouse cells is associated with elevated RAS activity and increased cell proliferation. Despite this straightforward pathophysiologic association between neuro®bromin, RAS, and tumorigenesis, recent insights from mouse and Drosophila modeling studies have suggested additional functions for neuro®bromin and have implicated Nf1 heterozygosity in tumor formation. Lastly, Nf1 knockout mouse studies have also demonstrated important roles for cooperating genetic changes that accelerate tumorigenesis as well as modi®er genes that impact on cancer susceptibility. Addresses Department of Neurology, Washington University School of Medicine, Box 8111; 660 S. Euclid Avenue, St. Louis, Missouri 63110, USA e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:20±27 This review comes from a themed issue on Oncogenes and cell proliferation Edited by Frank McCormick and Kevin Shannon 0959-437X/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(02)00015-1 Abbreviations cAMP cyclic adenosine monophosphate EGFR epidermal growth factor receptor GAP GTPase activating protein GM-CSF granulocyte-macrophage colony stimulating factor MAP mitogen-activated protein MPD myeloproliferative disease MPNST malignant peripheral nerve sheath tumor NF1 neuro®bromatosis type 1 NF1±GRD NF1±GAP-related domain PACAP38 pituitary adenylyl cyclase-activating polypeptide 38
Introduction
Neuro®bromatosis 1 (NF1) is one of the most common tumor predisposition syndromes affecting the nervous system [1]. Individuals with NF1 typically present in childhood with characteristic pigmentary abnormalities, including birthmarks (cafeÂ-au-lait macules), freckling in non-sun-exposed regions, and benign pigmented hamartomas of the iris (Lisch nodules). In addition, >50% of children with NF1 have speci®c learning disabilities and <10% will develop bony abnormalities, such as severe (dystrophic) scoliosis, orbital dysplasias, and bony defects of the arms and legs. Current Opinion in Genetics & Development 2003, 13:20±27
The most common tumor seen in individuals with NF1 is the neuro®broma, a benign growth associated with a peripheral nerve, which is composed of Schwann cells, ®broblasts, and mast cells [2]. Neuro®bromas typically appear in early adolescence as ¯eshy nodules either underneath or protruding from the skin. These tumors are always benign and never transform into malignant cancers. In contrast, over one-third of individuals with NF1 harbor a more diffuse neuro®broma tumor, termed a plexiform neuro®broma, that can undergo malignant transformation into a highly metastatic cancer (malignant peripheral nerve sheath tumor [MPNST]). MPNSTs are poorly responsive to conventional therapies and are frequently fatal. The second most common tumor in NF1 is the optic pathway glioma, a brain tumor composed of neoplastic astrocytes that is seen in 15±20% of children with NF1 [3]. Optic pathway gliomas are low-grade pilocytic astrocytomas that can cause blindness or endocrine abnormalities when involving the hypothalamus. These highly in®ltrative tumors are clinically less aggressive than histologically identical tumors in children without NF1. Lastly, young children with NF1 may infrequently develop myeloid leukemia and myelodysplastic syndrome [4]. With the identi®cation of the NF1 gene, considerable progress has been made in elucidating the molecular pathogenesis of speci®c NF1-associated clinical features. The purpose of our review is to present recent research progress highlighting insights gained from studies of Nf1de®cient mice and Drosophila.
Anatomy of the NF1 gene
The NF1 gene was identi®ed by positional cloning in 1990 and found to encode a large 220±250 kDa cytoplasmic protein product, neuro®bromin. Neuro®bromin is expressed in neurons, astrocytes, oligodendrocytes, Schwann cells, adrenal medullary cells, white blood cells, and gonadal tissue. Analysis of neuro®bromin revealed striking sequence similarity between a small central portion of the protein and members of the RAS GTPase-activating protein (RAS-GAP) family, including vertebrate p120-GAP, yeast ira1 and ira2, and Drosophila Gap1 (Figure 1a). NF1 comprises at least 60 exons with several alternatively spliced isoforms. One of the most common alternative splicing events involves exon 23a, which inserts an additional 21 amino acids into the NF1-GAP-related domain. In addition, several other isoforms have been detected that are differentially expressed in speci®c tissues, including brain neurons and muscle. www.current-opinion.com
Neurofibromatosis 1 Dasgupta and Gutmann 21
Figure 1
(a)
1
910
1172
Exon 23a
Domain related to yeast IRA
1538
2350
2818
NF1-GRD Receptor tyrosine kinase (RTK)
(b)
Neuropeptide receptor
RTK activation
GPCR
Receptor activation Heterotrimeric G proteins
Guanosine nucleotide exchange factors
Ras-GTP
GPCR activation
Ras-GDP ATP
cAMP
Neurofibromin RAF Adenylyl cyclase PI3 kinase
MEK PKA
cdc42 Rac1 Akt/PKB
Cell migration
Cell survival
Rac1
ERK
Rho
Gene expression
Cytoskeletal changes
Gene expression
Cell growth Learning Circadian rhythm and in mice in Drosophila proliferation
Learning and memory in Drosophila Current Opinion in Genetics & Development
Structure and function of neurofibromin. (a) The NF1 gene encodes a 2818 amino acid protein neurofibromin, which contains a 300 residue central region, termed the NF1±GRD. The region of neurofibromin containing residues 910±2350 shares sequence similarity with regions of yeast IRA proteins. The alternatively spliced exon 23a is contained within the NF1±GRD. (b) Neurofibromin accelerates inactivation of active GTP-bound RAS, such that NF1 loss in specific cells results in increased RAS activity and dysregulated cell growth. In addition, neurofibromin may also be required for cAMP- and protein kinase A (PKA)-mediated gene transcription.
Neuro®bromin function
On the basis of the predicted protein sequence, biochemical studies have con®rmed that neuro®bromin has RASGAP activity both in vitro and in vivo (Figure 1b). Loss of www.current-opinion.com
neuro®bromin in a wide variety of both human tumor and Nf1-de®cient mouse cells is associated with increased RAS activity and RAS effector (e.g. RAF, AKT and MAPK) activation [5,6]. In addition, direct inhibition of Current Opinion in Genetics & Development 2003, 13:20±27
22 Oncogenes and cell proliferation
RAS (protein farnesyltransferase inhibitors) or RAS pathway molecules (MEK and AKT inhibitors) as well as replacement of the NF1-GAP-related domain reverses the proliferative phenotype in Nf1-de®cient mouse cells [7,8]. These results strongly suggest that most of the growth advantage conferred by loss of neuro®bromin function results from dysregulated RAS activity. The RAS-GAP domain of neuro®bromin comprises only 10% of the entire polypeptide, which has raised the possibility that other regions of the molecule are important for modulating cell growth or other processes related to NF1-related disease. Initial studies in Drosophila have shown that neuro®bromin regulates G-protein-stimulated adenylate cyclase activity. Nf1 mutant ¯ies are smaller in body size and this phenotype can be rescued, not by attenuating RAS activity but by increasing cAMP production in response to overexpression of the cAMP-dependent protein kinase A [9]. In Drosophila, Nf1 is essential for the cellular response to neuropeptides, like PACAP38 (pituitary adenylyl cyclase-activating polypeptide 38), through activation of the adenylyl cyclase/cAMP pathway [10]. In mammals, PACAP38 has also been shown to induce cell growth in astrocytes and activate MAP kinase [11]. Although Nf1-de®cient murine primary neuronal cultures did not show any differences in morphology compared to wild-type cells, the cAMP concentration was signi®cantly lower in GTPgS-stimulated extracts of Nf1 / neurons [12]. These results suggest that some of the NF1 clinical abnormalities, such as short stature and learning disabilities, may result from other non-RAS neuro®bromin functions in speci®c cell types whereas other features, like tumor formation, involve hyperactivation of RAS. This point is underscored by recent studies in Drosophila demonstrating that circadian rhythm is altered in Nf1 mutant ¯ies but that this speci®c abnormality results from alterations in the RAS-MAP kinase signaling pathway [13]. Collectively, these ®ndings argue that defects in neuro®bromin function may affect different intracellular signaling pathways in particular cell types.
Lessons learned from the analysis of NF1-associated tumors
As neuro®bromas are complex heterogeneous tumors comprising multiple cell types, it was important for our understanding of the molecular pathogenesis of neuro®bromas to determine the cell of this tumor type and the contribution of the non-neoplastic cells to tumor development and progression. Several studies over the past few years have demonstrated at the DNA, RNA, and protein levels that the neoplastic cell of origin in neuro®bromas is the Schwann cell [14±16]. Moreover, loss of neuro®bromin in the Schwann cell, and not the ®broblast, is associated with increased RAS activity using a novel in vivo RAS-binding ¯uorescence assay [6]. The lack of RAS activation in neuro®broma ®broblasts suggests that neuCurrent Opinion in Genetics & Development 2003, 13:20±27
ro®bromin loss occurs only in the Schwann cell. These ®ndings also support the notion that NF1 inactivation in Schwann cells results in neoplastic Schwann cells critical for tumorigenesis but do not clarify the role of the ®broblasts and mast cells containing one mutant and one wild-type NF1 allele (NF1/ cells) to neuro®broma formation and progression (see section on `Conditional Nf1 knockout mice'). Plexiform neuro®bromas can undergo malignant transformation and form MPNSTs. Molecular studies have identi®ed MPNST-associated genetic alterations in a number of important mitogenic and cell-cycle regulatory pathways that synergize with NF1 loss and hyperactivation of RAS to promote transformation. Mutations and loss of heterozygosity involving the p53 tumor suppressor have been identi®ed in many NF1-associated as well as sporadic MPNSTs. In addition, mutations at the INK4 locus, which affect both p16INK4a and p14ARFtumor suppressor genes (see also review by Lowe and Sherr, this issue), have been found in NF1-associated MPNSTs [17]. Similarly, loss of another cell-cycle growth regulator, p27Kip1, has also been reported [18]. Collectively, these results suggest that the synergistic combination of increased mitogenic signaling that results from neuro®bromin loss and defective cell-cycle growth regulation may be suf®cient to initiate malignant transformation. Unexpectedly, DNA ampli®cation and increased protein expression of the epidermal growth factor receptor (EGF-R) has been found in MPNSTs. Activation of EGF-R by the EGF mitogen results in activation of RAS as well as other small signaling proteins. In the absence of neuro®bromin, augmented signaling through the EGF-R might have dramatic effects on cell proliferation and survival. In this regard, treatment of cultured Schwann cells derived from human MPNSTs with EGF-R antagonists blocked their proliferation [19]. In addition to MPNSTs, loss of heterozygosity at the NF1 locus is also observed in pheochromocytomas and myeloid leukemias [20]. In myeloid leukemias, the vast majority are associated with either loss of NF1 expression (individuals with NF1) or activating RAS mutations (sporadic leukemias) but not both [20,21]. These observations suggest that dysregulated RAS signaling Ð either resulting from activating RAS mutations or neuro®bromin loss Ð is central to the molecular pathogenesis of these cancers. Elegant studies on human NF1 leukemiaderived tumor cells [22] have shown that neuro®bromin loss results in hypersensitivity to granulocyte-macrophage colony stimulating factor (GM-CSF) stimulation, which promotes the survival, proliferation and differentiation of myeloid lineage cells. Studies of NF1-associated pilocytic astrocytomas have demonstrated loss of neuro®bromin expression [23] and www.current-opinion.com
Neurofibromatosis 1 Dasgupta and Gutmann 23
increased RAS pathway activation [24], whereas inactivation of NF1 is not observed in sporadic pilocytic astrocytomas [25]. Similarly, NF1-associated pilocytic astrocytomas lack the typical genetic changes observed in sporadic highgrade brain tumors [26]. These ®ndings collectively suggest that the genetic changes associated with progression of pilocytic astrocytomas in NF1 might be unique. Using gene-expression pro®ling, NF1-associated and sporadic pilocytic astrocytomas share a molecular signature with oligodendrocyte lineage cells, suggesting that the cell of origin of the pilocytic astrocytoma might be different from the conventional type 1 ®brillary astrocyte [27].
Lessons learned from animal models Conventional Nf1 knockout mice
Mouse models of NF1 have provided critical insights into the biology of human NF1 gene function (Figure 2). As is true for a large number of mammalian tumor suppressor genes, mice homozygous for a targeted mutation in Nf1 die in utero. These Nf1-de®cient embryos die between embryonic days 12.5 and 13.5 as a result of a cardiac vessel defect, termed double outlet right ventricle. These mice also exhibit neural tube closure defects (exencephaly) and endocardial cushion abnormalities [28].
Figure 2
(a)
p53/INK4/ARF mutations Schwann cell precursor
NF1 loss
Mature Schwann cell
NF1+/– mast cells, fibroblasts, perineurial cells
Plexiform neurofibroma
MPNST EGFR amplification
Dermal neurofibroma
NF1 loss
(b) Neural stem cells
NF1 loss Astrocytoma
Glial precursors NF1 LOH
p53 mutations
NF1+/– astroglia
Astrocytes
GBM
Increased cell proliferation, abnormal motility & adhesion
NF1–/– astrocytes
no tumor Increased cell proliferation, abnormal motility & adhesion
no tumor
(c)
NF1+/– hematopoietic precursor cells
NF1 LOH
Myeloid leukemia Current Opinion in Genetics & Development
Models of NF1-associated tumorigenesis. (a) NF1 loss in Schwann cell precursors during developmentÐin cooperation with NF1/ fibroblasts, perineurial cells, and mast cellsÐresults in plexiform neurofibroma formation. The accumulation of other genetic changes (e.g. p53, p16, or p27 loss) promotes transformation into MPNSTs. NF1 loss in Schwann cells in cooperation with NF1/ cells results in dermal neurofibroma formation. (b) Astroglial precursors following NF1 loss form astrocytomas that can progress with additional genetic changes to form high-grade glioblastoma multiforme tumors (GBM). These additional cooperating genetic alterations are uncommon in individuals with NF1. In contrast, NF1 loss in astrocytes alone is probably insufficient for tumor formation. (c) NF1 inactivation in myeloid precursors results in hypersensitivity to mitogenic cytokines, increased cell proliferation and myeloid leukemogenesis. LOH, loss of heterozygosity. www.current-opinion.com
Current Opinion in Genetics & Development 2003, 13:20±27
24 Oncogenes and cell proliferation
Nf1/ mice are cancer prone but do not develop the hallmark tumors seen in individuals with NF1 (neuro®bromas and astrocytomas). Instead these mice develop leukemia and pheochromocytoma, two tumor types occasionally seen in individuals with NF1. Approximately 10% of Nf1/ mice develop juvenile myelomonocytic leukemia and myeloproliferative disease (MPD) upon loss of the wild-type Nf1 allele in hematopoietic cells. In addition, MPD can be modeled in mice by the adoptive transfer of Nf1 / fetal liver cells. Nf1 / myeloid lineage cells are hypersensitive to GM-CSF, which promotes survival, proliferation and differentiation of myeloid lineage cells by activating the RAS-Raf-MAP kinase cascade [29]. In this regard, loss of neuro®bromin in hematopoietic precursors is suf®cient for myeloid leukemia and MPD, and GM-CSF hyperactivation of the RASsignaling pathway provides the mitogenic drive for leukemogenesis. Studies from our laboratory and others have demonstrated that Nf1 heterozygosity confers a biologic phenotype. Nf1/ mice exhibit increased numbers of brain astrocytes with abnormal attachment, spreading and motility properties [30,31] as well as a cell-autonomous growth advantage [5]. In addition, Nf1/ astrocytes demonstrate increased RAS pathway activation and a proliferative advantage that is primarily dependent on RAS [5]. Studies on Nf1/ mast and hematopoietic cells have also demonstrated a cell-autonomous growth advantage that re¯ects increased RAS pathway signaling. Both the increased proliferation and RAS pathway activation in these Nf1/ cells was reversed by the introduction of a wild-type NF1±GAP domain. Interestingly, no such effect was obtained with p120±GAP [7], emphasizing the critical and non-redundant role of neuro®bromin in RAS regulation. Lastly, in Nf1/ hematopoietic cells, the increased proliferation observed in response to speci®c mitogens is partly because of Rac2-mediated cross-talk between the PI3 kinase and RAS±Raf±MEK±ERK pathways [32].
teriophage Cre recombinase-mediated excision and inactivation of a targeted Nf1 gene containing intronic recombinatorial LoxP sites. Mice harboring Nf1 alleles with LoxP sequences (Nf1¯ox/¯ox mice) express neuro®bromin and are phenotypically normal in the absence of Cre expression. Intercrossing of Nf1¯ox/¯ox mice with transgenic strains expressing Cre recombinase under the control of tissue-speci®c promoters results in Nf1 inactivation in speci®c tissues. Using this approach, Nf1 has been inactivated speci®cally in neurons, Schwann cells and astrocytes. Neuron-speci®c Nf1 inactivation resulted in mice that demonstrate neurotrophin-independent dorsal root ganglion neuron survival in vitro and an overall reduction in brain cortical thickness. Aberrant neuronal survival was also observed in vitro, associated with increased cellularity and astrogliosis throughout cortex, hippocampus and brainstem [34]. Schwann-cell-speci®c Nf1 conditional knockout mice develop prominent Schwann-cell hyperplasia but few tumors [35]. Schwann-cell-speci®c Nf1 knockout mice generated to express one mutant and one wild-type allele in all cells (Nf1¯ox/mut; Krox20±Cre mice) develop plexiform neuro®broma-like tumors along peripheral nerves. Examination of the neuro®bromas in these mice demonstrates in®ltration of Nf1/ mast cells, suggesting that Nf1/ cells in the tumor environment may promote tumorigenesis. These ®ndings concur with previous mouse modeling experiments in which chimeric animals were generated by injecting Nf1 / embryonic stem cells into blastocyst-stage embryos. Only those embryos containing a small minority of Nf1 / cells survived to adulthood. Most of these mice developed multiple neuro®bromas reminiscent of plexiform neuro®bromas, suggesting that other neuro®bromin-de®cient cell types (e.g. mast cells) contributed to the development of these tumors [36].
In addition to growth-regulation defects, Nf1/ mice exhibit speci®c de®cits in learning and spatial memory, which likely re¯ects increased RAS activity and impairment of long-term potentiation. These Nf1/ learning abnormalities can be rescued by either genetic or pharmacologic manipulations of RAS activity or GABA receptor antagonists [33].
Astrocyte-speci®c Nf1 conditional knockout mice exhibit increased astrocyte proliferation both in vitro and in vivo, but do not develop gliomas even after 20 months of age [37]. In these studies, Nf1 inactivation in astrocytes by embryonic day 14 is not suf®cient for astrocytoma formation. These observations raise the intriguing possibility that either the cell of origin of NF1-associated gliomas is not the conventional glial ®brillary acidic protein-expressing astrocyte or that additional genetic changes are required.
Conditional Nf1 knockout mice
Cooperating genetic changes
The early embryonic lethal phenotype of the Nf1 knockout precludes a detailed analysis of the contribution of Nf1 to neuro®broma and astrocytoma formation. For this reason, Parada and co-workers have developed conditional knockout mice to facilitate the study of speci®c tissues lacking neuro®bromin expression [34]. Conditional Nf1 knockout mice result from tissue-speci®c bacCurrent Opinion in Genetics & Development 2003, 13:20±27
Another approach to studying NF1 tumor formation involves the generation of mice heterozygous for mutations in both Nf1 and other relevant tumor suppressor genes. On the basis of genetic studies of NF1-associated MPNSTs implicating the p53 tumor suppressor, investigators have generated Nf1/ ; p53/ mice [38]. These double heterozygote mice developed high-grade sarcowww.current-opinion.com
Neurofibromatosis 1 Dasgupta and Gutmann 25
mas with histopathologic features of human MPNST [38], demonstrating that loss of both neuro®bromin and p53 cooperate to facilitate MPNST formation. In a related study [39], 60% of p19ARF null mice generated on an Nf1/ background developed multiple tumors (sarcomas, lymphomas, leukemias and carcinomas), suggesting genetic cooperativity between these two tumor suppressor proteins in tumorigenesis. In addition, there are likely modifying genes that specify tumor susceptibility as well as the spectrum of speci®c tumors, as demonstrated using mice harboring both Nf1 and p53 mutations [40]. In this elegant study, Nf1/ ; p53/ mice developed highly aggressive glioblastoma multiforme brain tumors when maintained on the C57BL/6, compared to the SJL/J, genetic background. Aberrant EGFR signaling has also been observed in NF1-associated MPNSTs. Although EGFR is not normally expressed in Schwann cells, EGFR overexpression and signaling has been detected in MPNSTs from NF1 patients as well as transformed Nf1 / Schwann cells [19]. In addition, cell lines derived from mice heterozygous for mutations in both Nf1 and p53 overexpress EGFR and activate the PI3K-AKT pathway in response to EGFR stimulation [41].
Exon-speci®c Nf1 knockout mice
The existence of multiple neuro®bromin isoforms has raised the interesting possibility that speci®c isoforms have unique functions beyond RAS regulation. In an effort to address this hypothesis, Silva and co-workers have generated mice lacking exon 23a (involving the NF1±GAP-related domain [NF1-GRD]) and the brainspeci®c exon 9a (Y Elgersma, A Silva, personal communication). Targeted disruption of exon 23a resulted in learning impairments in mice, similar to conventional Nf1/ mice [42]. It is possible that the exon 23a knockout mice develop learning disabilities, not as a result of impaired RAS-GAP activity but rather because of reduced neuro®bromin activity attributable to exon 23a function, such as cAMP regulation. Further studies on exon 23a knockout mice may provide insights into the relationship between neuro®bromin and learning. The fact that these mice did not develop tumors likewise suggests that other functions of neuro®bromin attributable to the NF1±GRD may be important for growth regulation.
Conclusions
Signi®cant insights into the molecular pathogenesis of NF1-associated tumors have derived from studies of both human tumors and Nf1 knockout mice. The complementary information that results from these different approaches has raised interesting issues regarding the genetic and biological factors that contribute to tumorigenesis in individuals with NF1. It is clear that NF1 inactivation is suf®cient for the formation of some tumors www.current-opinion.com
in NF1, such as pheochromocytomas and leukemias, but is not suf®cient by itself to result in neuro®bromas, astrocytomas or MPNSTs. Whereas neuro®bromas require other participating Nf1/ cells to provide the proper cellular or mitogenic environment for tumor formation, astrocytoma formation may require Nf1 inactivation in speci®c astroglial progenitor cell populations or the presence of additional cooperating genetic changes. The notion that Nf1/ cells are not the biological equivalent of wild-type cells expands our appreciation of the role of tumor suppressor gene heterozygosity in growth control and tumorigenesis. It is highly likely that some of the clinical features of NF1 result solely from NF1 heterozygosity. As we enter into an era of targeted cancer therapeutics, an improved understanding of the signaling pathways dysregulated as a consequence of neuro®bromin loss will provide additional molecular targets for drug design. The future of clinical care for individuals with NF1 could one day involve drugs with highly selective activity towards speci®c biochemical pathways deranged in different cell types responsible for particular clinical features of NF1. Further work on the precise role of neuro®bromin in RAS and cAMP pathway regulation in speci®c cell types in both model organisms and human tissues may provide these essential insights.
References and recommended reading
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The I, Hannigan GE, Cowley GS, Reginald S, Zhong Y, Gusella JF, Hariharan IK: Bernards A: Rescue of Drosophila NF1 mutant phenotype by protein kinase A. Science 1997, 276:791-794.
10. Guo HF, The I, Hannan F, Bernards A, Zhong Y: Requirement of Drosophila NF1 for activation of adenylyl cyclase by PACAP-like neuropeptides. Science 1997, 276:795-798. 11. Moroo I, Tatsuno I, Uchida D, Tanaka T, Saito J, Saito Y, Hirai A: Pituitary adenylate cyclase activating peptide (PACAP) stimulates mitogen-activated protein kinase (MAPK) in cultured rat astrocytes. Brain Res 1998, 795:191-196. 12. Tong J, Hannan F, Zhu Y, Bernards A, Zhong Y: Neuro®bromin regulates G protein- stimulated adenylyl cyclase activity. Nat Neurosci 2002, 5:95-96. This report provides the ®rst direct evidence that neuro®bromin loss in mammalian cells results in cAMP pathway dysfunction. In this study, the authors demonstrate that G-protein-stimulated adenylyl cyclase activity was lower in Nf1 / than in to Nf1/ mouse neurons, supporting the notion that neuro®bromin has additional functions in mammalian cells besides RAS regulation. 13. Williams JA, Su HS, Bernards A, Field J, Sehgal A: A circadian output in Drosophila mediated by neuro®bromatosis-1 and Ras/MAPK. Science 2001, 293:2251-2256. This report describes a RAS-mediated physiologic abnormality in Drosophila that is not related to cAMP/PKA regulation. In this study, the authors demonstrate that circadian rhythms are defective in Nf1 / Drosophila, but that this abnormality results from dysregulated RAS activity. These ®ndings support the notion that neuro®bromin may have both RAS-dependent and RAS-independent functions in Drosophila. 14. Rutkowski JL, Wu K, Gutmann DH, Boyer PJ, Legius E: Genetic and cellular defects contributing to benign tumor formation in neuro®bromatosis type 1. Hum Mol Genet 2000, 12:1059-1066. 15. Muir D, Neubauer D, Lim IT, Yachnis AT, Wallace MR: Tumorigenic properties of neuro®bromin-de®cient neuro®broma Schwann cells. Am J Pathol 2001, 158:501-513. 16. Perry A, Roth KA, Banerjee R, Fuller CE, Gutmann DH: NF1 deletions in S-100 protein positive and negative cells of sporadic and neuro®bromatosis 1 (NF1)-associates plexiform neuro®bromas and malignant peripheral nerve sheath tumors. Am J Pathol 2001, 159:57-61. 17. Kourea HP, Orlow I, Scheithauer BW, Cordon-Cardo C, Woodruff JM: Deletions of the INK4A gene occur in malignant peripheral nerve sheath tumors but not in neuro®bromas. Am J Pathol 1999, 155:1855-1860. 18. Kourea HP, Cordon-Cardo C, Dudas M, Leung D, Woodruff JM: Expression of p27 (kip) and other cell cycle regulators in malignant peripheral nerve sheath tumors and neuro®bromas: the emerging role of p27 (kip) in malignant transformation of neuro®bromas. Am J Pathol 1999, 155:1885-1891. 19. DeClue JE, Heffel®nger S, Benvenuto G, Ling B, Li S, Rui W, Vass WC, Viskochil D, Ratner N: Epidermal growth factor expression in neuro®bromatosis type1-related tumors and NF1 animal models. J Clin Invest 2000, 105:1233-1241. 20. Side L, Taylor B, Cayouette M, Conner E, Thompson P, Luce M, Shannon K: Homozygous inactivation of the NF1 gene in bone marrow cells from children with neuro®bromatosis type 1 and malignant myeloid disorders. N Eng J Med 1997, 336:1713-1720. 21. Side LE, Emanuel PD, Taylor B, Franklin J, Thompson P, Castleberry RP, Shannon KM: Mutations of the NF1 gene in children with juvenile myelomonocytic leukemia without clinical evidence of neuro®bromatosis, type 1. Blood 1998, 92:267-272. 22. Birnbaum RA, O'Marcaigh A, Wardak Z, Zhang YY, Dranoff G, Jacks T, Clapp DW, Shannon KM: NF1 and Gmcsf interact in myeloid leukemogenesis. Mol Cell 2000, 5:189-195. 23. Gutmann DH, Donahoe J, Brown T, James CD, Perry A: Loss of neuro®bromatosis 1 (NF1) gene expression in NF1-associated pilocytic astrocytomas. Neuropathol Appl Neurobiol 2000, 26:361-367. 24. Lau N, Feldkamp MM, Roncari L, Loehr AH, Shannon P, Gutmann DH, Guha A: Loss of neuro®bromin is associated with activation of Ras/MAPK and PI3K/AKT signaling in a neuro®bromatosis 1 astrocytoma. J Neuropathol Exp Neurol 2000, 59:759-776. Current Opinion in Genetics & Development 2003, 13:20±27
25. Kluwe L, Hagel C, Tatagiba M, Thomas S, Stavrou D, Ostertag H, von Deimling A, Mautner VF: Loss of NF1 alleles distinguish sporadic from NF1-associated pilocytic astrocytomas. J Neuropathol Exp Neurol 2001, 60:917-920. 26. Li J, Perry A, James CD, Gutmann DH: Cancer-related gene expression pro®les in NF1-associated pilocytic astrocytomas. Neurology 2001, 56:885-890. 27. Gutmann DH, Hedrick NM, Li J, Nagarajan R, Perry A, Watson MA: Comparative gene expression pro®le analysis of Neuro®bromatosis1-associated and sporadic pilocytic astrocytomas. Cancer Res 2002, 62:2085-2091. 28. Lakkis MM, Golden JA, O'Shea S, Epstein JA: Neuro®bromin de®ciency in mice causes excencephaly and is a modi®er for Splotch neural tube defects. Dev Biol 1999, 212:80-92. 29. Zhang Y, Vik TA, Ryder JW, Srour EF, Jacks T, Shannon K, Clapp DW: Nf1 regulates hematopoietic progenitor cell growth and Ras signalling in response to multiple cytokines. J Exp Med 1998, 187:1893-1902. 30. Gutmann DH, Loehr A, Zhang Y, Kim J, Henkemeyer M, Cashes A: Haploinsuf®ciency for neuro®bromatosis 1 (NF1) tumor suppressor results in increased astrocyte proliferation. Oncogene 1999, 18:4450-4459. 31. Gutmann DH, Wu YL, Hedrick NM, Zhu Y, Guha A, Parada LF: Heterozygosity for neuro®bromatosis 1 (NF1) tumor suppressor results in abnormalities in cell attachment, spreading and motility in astrocytes. Hum Mol Genet 2001, 10:3009-30016. These two reports [30,31] describe a direct effect of Nf1 heterozygosity on astrocyte proliferation and actin cytoskeleton-mediated processes. In these studies, the authors demonstrate that Nf1/ astrocytes exhibit increased cell proliferation, alterations in actin cytoskeleton-mediated processes, and dysregulated RAS activity. These observations support the notion that Nf1/ cells are not functionally equivalent to wild-type cells and may contribute to the pathogenesis of NF1-associated features. 32. Ingram DA, Hiatt K, King AJ, Fisher L, Shivakumar R, Christina D, Wenning MJ, Bruce D, Travers JB, Hood A et al.: Hyperactivation of p21ras and hematopoietic-speci®c Rho GTPase, Rac2, cooperate to alter the proliferation of neuro®bromin-de®cient mast cells in vivo and in vitro. J Exp Med 2001, 194:57-69. 33. Costa RM, Federov NB, Kogan JH, Murphu GG, Stern J, Ohno M, Kucherlapati R, Jacks T, Silva A: Mechanism for the learning de®cits in a mouse model of neuro®bromatosis type 1. Nature 2002, 415:526-530. 34. Zhu Y, Romero MI, Ghosh P, Ye Z, Charnay P, Rushing EJ, Marsh JD, Parada LF: Ablation of NF1 functions in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Genes Dev 2001, 15:859-876. 35. Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF: Neuro®bromas in NF1: Schwann cell origin and role of tumor environment. Science 2002, 296:920-922. This report describes the requirement for contributing Nf1/ cells to plexiform neuro®broma formation in mice lacking neuro®bromin in Schwann cells. This landmark study demonstrates for the ®rst time that neuro®bromin loss in Schwann cells by itself may not be suf®cient for tumorigenesis and that plexiform neuro®broma formation requires the contribution of Nf1/ cells (e.g. mast cells). 36. Cichowski K, Shih TS, Schmitt E, Santiago S, Reilly K, McLaughlin ME, Roderick TB, Bronson RT, Jacks T: Mouse models of tumor development in neuro®bromatosis type 1. Science 1999, 286:2172-2176. 37. Bajenaru ML, Zhu Y, Hedrick NM, Donahoe J, Parada LF, Gutmann DH: Astrocyte-speci®c inactivation of the neuro®bromatosis 1 gene (NF1) is insuf®cient for astrocytoma formation. Mol Cell Biol 2002, 22:5100-5113. This report demonstrates that neuro®bromin loss in maturing astrocytes is not suf®cient for astrocytoma formation in the mouse. In this study, the authors employ multiple approaches to inactivating neuro®bromin in astrocytes and ®nd that Nf1 loss in type-1 astrocytes does not confer a neoplastic phenotype. These ®ndings suggest that NF1-associated glioma formation may require additional cellular or genetic changes. 38. Vogel KS, Kleese LJ, Velasco-Miguel S, Meyers K, Rushing EJ, Parada LF: Mouse tumor model of neuro®bromatosis type 1. Science 1999, 286:2176-2179. www.current-opinion.com
Neurofibromatosis 1 Dasgupta and Gutmann 27
39. King D, Yang G, Thompson MA, Hiebert SW: Loss of neuro®bromatosis-1 and p19ARF cooperate to induce multiple tumor phenotype. Oncogene 2002, 21:4978-4982. 40. Reilly KM, Loisel DA, Bronson RT, McLaughlin ME, Jacks T: Nf1; Trp53 mice develop glioblastoma with evidence of strain-speci®c effects. Nat Genet 2000, 26:109-113. This report demonstrates that the genetic background has a signi®cant effect on tumor formation as well as the types of tumors generated in mice. In this study, the authors report that Nf1/ ; p53/ mice maintained on speci®cgenetic backgrounds develop brain tumorswhereasinotherstrains, brain tumor development is uncommon. These ®ndings suggest that additional `modi®er' genes exist that in¯uence tumorigenesis in the mouse. 41. Li H, Velasco-Miguel V, Vass CW, Parada LF, DeClue JE: Epidermal growth factor receptor signaling pathways are associated with tumorigenesis in the Nf1:p53 mouse tumor model. Cancer Res 2002, 62:4507-4513.
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This report describes the cooperative effect of EGFR expression and activation in Schwann cell tumor proliferation in mice developing MPNST. The authors ®nd that EGFR overexpression is seen in both mouse and human MPNSTs, suggesting that additional signaling abnormalities conferred by the aberrant expression of EGFR impact on the pathogenesis of one of the malignant tumor types in NF1. 42. Costa RM, Yang T, Huynh DP, Pulst SM, Viskochil DH, Silva AJ, Brannan CI: Learning de®cits, but normal development and tumor predisposition, in mice lacking exon 23a of Nf1. Nat Genet 2001, 27:399-405. This report describes the differential effects of exon 23a deletion on mouse development, tumor formation, and learning. In this study, the authors generate mice lacking exon 23a and demonstrate that these mice exhibit learning de®cits similar to Nf1/ mice, but are not prone to tumors. These ®ndings suggest that neuro®bromin-containing exon 23a contributes to learning and memory in mice, but has a limited role in tumor formation.
Current Opinion in Genetics & Development 2003, 13:20±27
Neuro®bromatosis 1: closing the GAP between mice and men Biplab Dasgupta and David H Gutmann Neuro®bromatosis 1 (NF1) is a common genetic condition in which affected individuals are prone to the development of benign and malignant tumors. The NF1 tumor suppressor encodes a protein product, neuro®bromin, which functions in part as a negative regulator of RAS. Loss of neuro®bromin expression in NF1-associated tumors or Nf1-de®cient mouse cells is associated with elevated RAS activity and increased cell proliferation. Despite this straightforward pathophysiologic association between neuro®bromin, RAS, and tumorigenesis, recent insights from mouse and Drosophila modeling studies have suggested additional functions for neuro®bromin and have implicated Nf1 heterozygosity in tumor formation. Lastly, Nf1 knockout mouse studies have also demonstrated important roles for cooperating genetic changes that accelerate tumorigenesis as well as modi®er genes that impact on cancer susceptibility. Addresses Department of Neurology, Washington University School of Medicine, Box 8111; 660 S. Euclid Avenue, St. Louis, Missouri 63110, USA e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:20±27 This review comes from a themed issue on Oncogenes and cell proliferation Edited by Frank McCormick and Kevin Shannon 0959-437X/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(02)00015-1 Abbreviations cAMP cyclic adenosine monophosphate EGFR epidermal growth factor receptor GAP GTPase activating protein GM-CSF granulocyte-macrophage colony stimulating factor MAP mitogen-activated protein MPD myeloproliferative disease MPNST malignant peripheral nerve sheath tumor NF1 neuro®bromatosis type 1 NF1±GRD NF1±GAP-related domain PACAP38 pituitary adenylyl cyclase-activating polypeptide 38
Introduction
Neuro®bromatosis 1 (NF1) is one of the most common tumor predisposition syndromes affecting the nervous system [1]. Individuals with NF1 typically present in childhood with characteristic pigmentary abnormalities, including birthmarks (cafeÂ-au-lait macules), freckling in non-sun-exposed regions, and benign pigmented hamartomas of the iris (Lisch nodules). In addition, >50% of children with NF1 have speci®c learning disabilities and <10% will develop bony abnormalities, such as severe (dystrophic) scoliosis, orbital dysplasias, and bony defects of the arms and legs. Current Opinion in Genetics & Development 2003, 13:20±27
The most common tumor seen in individuals with NF1 is the neuro®broma, a benign growth associated with a peripheral nerve, which is composed of Schwann cells, ®broblasts, and mast cells [2]. Neuro®bromas typically appear in early adolescence as ¯eshy nodules either underneath or protruding from the skin. These tumors are always benign and never transform into malignant cancers. In contrast, over one-third of individuals with NF1 harbor a more diffuse neuro®broma tumor, termed a plexiform neuro®broma, that can undergo malignant transformation into a highly metastatic cancer (malignant peripheral nerve sheath tumor [MPNST]). MPNSTs are poorly responsive to conventional therapies and are frequently fatal. The second most common tumor in NF1 is the optic pathway glioma, a brain tumor composed of neoplastic astrocytes that is seen in 15±20% of children with NF1 [3]. Optic pathway gliomas are low-grade pilocytic astrocytomas that can cause blindness or endocrine abnormalities when involving the hypothalamus. These highly in®ltrative tumors are clinically less aggressive than histologically identical tumors in children without NF1. Lastly, young children with NF1 may infrequently develop myeloid leukemia and myelodysplastic syndrome [4]. With the identi®cation of the NF1 gene, considerable progress has been made in elucidating the molecular pathogenesis of speci®c NF1-associated clinical features. The purpose of our review is to present recent research progress highlighting insights gained from studies of Nf1de®cient mice and Drosophila.
Anatomy of the NF1 gene
The NF1 gene was identi®ed by positional cloning in 1990 and found to encode a large 220±250 kDa cytoplasmic protein product, neuro®bromin. Neuro®bromin is expressed in neurons, astrocytes, oligodendrocytes, Schwann cells, adrenal medullary cells, white blood cells, and gonadal tissue. Analysis of neuro®bromin revealed striking sequence similarity between a small central portion of the protein and members of the RAS GTPase-activating protein (RAS-GAP) family, including vertebrate p120-GAP, yeast ira1 and ira2, and Drosophila Gap1 (Figure 1a). NF1 comprises at least 60 exons with several alternatively spliced isoforms. One of the most common alternative splicing events involves exon 23a, which inserts an additional 21 amino acids into the NF1-GAP-related domain. In addition, several other isoforms have been detected that are differentially expressed in speci®c tissues, including brain neurons and muscle. www.current-opinion.com
Neurofibromatosis 1 Dasgupta and Gutmann 21
Figure 1
(a)
1
910
1172
Exon 23a
Domain related to yeast IRA
1538
2350
2818
NF1-GRD Receptor tyrosine kinase (RTK)
(b)
Neuropeptide receptor
RTK activation
GPCR
Receptor activation Heterotrimeric G proteins
Guanosine nucleotide exchange factors
Ras-GTP
GPCR activation
Ras-GDP ATP
cAMP
Neurofibromin RAF Adenylyl cyclase PI3 kinase
MEK PKA
cdc42 Rac1 Akt/PKB
Cell migration
Cell survival
Rac1
ERK
Rho
Gene expression
Cytoskeletal changes
Gene expression
Cell growth Learning Circadian rhythm and in mice in Drosophila proliferation
Learning and memory in Drosophila Current Opinion in Genetics & Development
Structure and function of neurofibromin. (a) The NF1 gene encodes a 2818 amino acid protein neurofibromin, which contains a 300 residue central region, termed the NF1±GRD. The region of neurofibromin containing residues 910±2350 shares sequence similarity with regions of yeast IRA proteins. The alternatively spliced exon 23a is contained within the NF1±GRD. (b) Neurofibromin accelerates inactivation of active GTP-bound RAS, such that NF1 loss in specific cells results in increased RAS activity and dysregulated cell growth. In addition, neurofibromin may also be required for cAMP- and protein kinase A (PKA)-mediated gene transcription.
Neuro®bromin function
On the basis of the predicted protein sequence, biochemical studies have con®rmed that neuro®bromin has RASGAP activity both in vitro and in vivo (Figure 1b). Loss of www.current-opinion.com
neuro®bromin in a wide variety of both human tumor and Nf1-de®cient mouse cells is associated with increased RAS activity and RAS effector (e.g. RAF, AKT and MAPK) activation [5,6]. In addition, direct inhibition of Current Opinion in Genetics & Development 2003, 13:20±27
22 Oncogenes and cell proliferation
RAS (protein farnesyltransferase inhibitors) or RAS pathway molecules (MEK and AKT inhibitors) as well as replacement of the NF1-GAP-related domain reverses the proliferative phenotype in Nf1-de®cient mouse cells [7,8]. These results strongly suggest that most of the growth advantage conferred by loss of neuro®bromin function results from dysregulated RAS activity. The RAS-GAP domain of neuro®bromin comprises only 10% of the entire polypeptide, which has raised the possibility that other regions of the molecule are important for modulating cell growth or other processes related to NF1-related disease. Initial studies in Drosophila have shown that neuro®bromin regulates G-protein-stimulated adenylate cyclase activity. Nf1 mutant ¯ies are smaller in body size and this phenotype can be rescued, not by attenuating RAS activity but by increasing cAMP production in response to overexpression of the cAMP-dependent protein kinase A [9]. In Drosophila, Nf1 is essential for the cellular response to neuropeptides, like PACAP38 (pituitary adenylyl cyclase-activating polypeptide 38), through activation of the adenylyl cyclase/cAMP pathway [10]. In mammals, PACAP38 has also been shown to induce cell growth in astrocytes and activate MAP kinase [11]. Although Nf1-de®cient murine primary neuronal cultures did not show any differences in morphology compared to wild-type cells, the cAMP concentration was signi®cantly lower in GTPgS-stimulated extracts of Nf1 / neurons [12]. These results suggest that some of the NF1 clinical abnormalities, such as short stature and learning disabilities, may result from other non-RAS neuro®bromin functions in speci®c cell types whereas other features, like tumor formation, involve hyperactivation of RAS. This point is underscored by recent studies in Drosophila demonstrating that circadian rhythm is altered in Nf1 mutant ¯ies but that this speci®c abnormality results from alterations in the RAS-MAP kinase signaling pathway [13]. Collectively, these ®ndings argue that defects in neuro®bromin function may affect different intracellular signaling pathways in particular cell types.
Lessons learned from the analysis of NF1-associated tumors
As neuro®bromas are complex heterogeneous tumors comprising multiple cell types, it was important for our understanding of the molecular pathogenesis of neuro®bromas to determine the cell of this tumor type and the contribution of the non-neoplastic cells to tumor development and progression. Several studies over the past few years have demonstrated at the DNA, RNA, and protein levels that the neoplastic cell of origin in neuro®bromas is the Schwann cell [14±16]. Moreover, loss of neuro®bromin in the Schwann cell, and not the ®broblast, is associated with increased RAS activity using a novel in vivo RAS-binding ¯uorescence assay [6]. The lack of RAS activation in neuro®broma ®broblasts suggests that neuCurrent Opinion in Genetics & Development 2003, 13:20±27
ro®bromin loss occurs only in the Schwann cell. These ®ndings also support the notion that NF1 inactivation in Schwann cells results in neoplastic Schwann cells critical for tumorigenesis but do not clarify the role of the ®broblasts and mast cells containing one mutant and one wild-type NF1 allele (NF1/ cells) to neuro®broma formation and progression (see section on `Conditional Nf1 knockout mice'). Plexiform neuro®bromas can undergo malignant transformation and form MPNSTs. Molecular studies have identi®ed MPNST-associated genetic alterations in a number of important mitogenic and cell-cycle regulatory pathways that synergize with NF1 loss and hyperactivation of RAS to promote transformation. Mutations and loss of heterozygosity involving the p53 tumor suppressor have been identi®ed in many NF1-associated as well as sporadic MPNSTs. In addition, mutations at the INK4 locus, which affect both p16INK4a and p14ARFtumor suppressor genes (see also review by Lowe and Sherr, this issue), have been found in NF1-associated MPNSTs [17]. Similarly, loss of another cell-cycle growth regulator, p27Kip1, has also been reported [18]. Collectively, these results suggest that the synergistic combination of increased mitogenic signaling that results from neuro®bromin loss and defective cell-cycle growth regulation may be suf®cient to initiate malignant transformation. Unexpectedly, DNA ampli®cation and increased protein expression of the epidermal growth factor receptor (EGF-R) has been found in MPNSTs. Activation of EGF-R by the EGF mitogen results in activation of RAS as well as other small signaling proteins. In the absence of neuro®bromin, augmented signaling through the EGF-R might have dramatic effects on cell proliferation and survival. In this regard, treatment of cultured Schwann cells derived from human MPNSTs with EGF-R antagonists blocked their proliferation [19]. In addition to MPNSTs, loss of heterozygosity at the NF1 locus is also observed in pheochromocytomas and myeloid leukemias [20]. In myeloid leukemias, the vast majority are associated with either loss of NF1 expression (individuals with NF1) or activating RAS mutations (sporadic leukemias) but not both [20,21]. These observations suggest that dysregulated RAS signaling Ð either resulting from activating RAS mutations or neuro®bromin loss Ð is central to the molecular pathogenesis of these cancers. Elegant studies on human NF1 leukemiaderived tumor cells [22] have shown that neuro®bromin loss results in hypersensitivity to granulocyte-macrophage colony stimulating factor (GM-CSF) stimulation, which promotes the survival, proliferation and differentiation of myeloid lineage cells. Studies of NF1-associated pilocytic astrocytomas have demonstrated loss of neuro®bromin expression [23] and www.current-opinion.com
Neurofibromatosis 1 Dasgupta and Gutmann 23
increased RAS pathway activation [24], whereas inactivation of NF1 is not observed in sporadic pilocytic astrocytomas [25]. Similarly, NF1-associated pilocytic astrocytomas lack the typical genetic changes observed in sporadic highgrade brain tumors [26]. These ®ndings collectively suggest that the genetic changes associated with progression of pilocytic astrocytomas in NF1 might be unique. Using gene-expression pro®ling, NF1-associated and sporadic pilocytic astrocytomas share a molecular signature with oligodendrocyte lineage cells, suggesting that the cell of origin of the pilocytic astrocytoma might be different from the conventional type 1 ®brillary astrocyte [27].
Lessons learned from animal models Conventional Nf1 knockout mice
Mouse models of NF1 have provided critical insights into the biology of human NF1 gene function (Figure 2). As is true for a large number of mammalian tumor suppressor genes, mice homozygous for a targeted mutation in Nf1 die in utero. These Nf1-de®cient embryos die between embryonic days 12.5 and 13.5 as a result of a cardiac vessel defect, termed double outlet right ventricle. These mice also exhibit neural tube closure defects (exencephaly) and endocardial cushion abnormalities [28].
Figure 2
(a)
p53/INK4/ARF mutations Schwann cell precursor
NF1 loss
Mature Schwann cell
NF1+/– mast cells, fibroblasts, perineurial cells
Plexiform neurofibroma
MPNST EGFR amplification
Dermal neurofibroma
NF1 loss
(b) Neural stem cells
NF1 loss Astrocytoma
Glial precursors NF1 LOH
p53 mutations
NF1+/– astroglia
Astrocytes
GBM
Increased cell proliferation, abnormal motility & adhesion
NF1–/– astrocytes
no tumor Increased cell proliferation, abnormal motility & adhesion
no tumor
(c)
NF1+/– hematopoietic precursor cells
NF1 LOH
Myeloid leukemia Current Opinion in Genetics & Development
Models of NF1-associated tumorigenesis. (a) NF1 loss in Schwann cell precursors during developmentÐin cooperation with NF1/ fibroblasts, perineurial cells, and mast cellsÐresults in plexiform neurofibroma formation. The accumulation of other genetic changes (e.g. p53, p16, or p27 loss) promotes transformation into MPNSTs. NF1 loss in Schwann cells in cooperation with NF1/ cells results in dermal neurofibroma formation. (b) Astroglial precursors following NF1 loss form astrocytomas that can progress with additional genetic changes to form high-grade glioblastoma multiforme tumors (GBM). These additional cooperating genetic alterations are uncommon in individuals with NF1. In contrast, NF1 loss in astrocytes alone is probably insufficient for tumor formation. (c) NF1 inactivation in myeloid precursors results in hypersensitivity to mitogenic cytokines, increased cell proliferation and myeloid leukemogenesis. LOH, loss of heterozygosity. www.current-opinion.com
Current Opinion in Genetics & Development 2003, 13:20±27
24 Oncogenes and cell proliferation
Nf1/ mice are cancer prone but do not develop the hallmark tumors seen in individuals with NF1 (neuro®bromas and astrocytomas). Instead these mice develop leukemia and pheochromocytoma, two tumor types occasionally seen in individuals with NF1. Approximately 10% of Nf1/ mice develop juvenile myelomonocytic leukemia and myeloproliferative disease (MPD) upon loss of the wild-type Nf1 allele in hematopoietic cells. In addition, MPD can be modeled in mice by the adoptive transfer of Nf1 / fetal liver cells. Nf1 / myeloid lineage cells are hypersensitive to GM-CSF, which promotes survival, proliferation and differentiation of myeloid lineage cells by activating the RAS-Raf-MAP kinase cascade [29]. In this regard, loss of neuro®bromin in hematopoietic precursors is suf®cient for myeloid leukemia and MPD, and GM-CSF hyperactivation of the RASsignaling pathway provides the mitogenic drive for leukemogenesis. Studies from our laboratory and others have demonstrated that Nf1 heterozygosity confers a biologic phenotype. Nf1/ mice exhibit increased numbers of brain astrocytes with abnormal attachment, spreading and motility properties [30,31] as well as a cell-autonomous growth advantage [5]. In addition, Nf1/ astrocytes demonstrate increased RAS pathway activation and a proliferative advantage that is primarily dependent on RAS [5]. Studies on Nf1/ mast and hematopoietic cells have also demonstrated a cell-autonomous growth advantage that re¯ects increased RAS pathway signaling. Both the increased proliferation and RAS pathway activation in these Nf1/ cells was reversed by the introduction of a wild-type NF1±GAP domain. Interestingly, no such effect was obtained with p120±GAP [7], emphasizing the critical and non-redundant role of neuro®bromin in RAS regulation. Lastly, in Nf1/ hematopoietic cells, the increased proliferation observed in response to speci®c mitogens is partly because of Rac2-mediated cross-talk between the PI3 kinase and RAS±Raf±MEK±ERK pathways [32].
teriophage Cre recombinase-mediated excision and inactivation of a targeted Nf1 gene containing intronic recombinatorial LoxP sites. Mice harboring Nf1 alleles with LoxP sequences (Nf1¯ox/¯ox mice) express neuro®bromin and are phenotypically normal in the absence of Cre expression. Intercrossing of Nf1¯ox/¯ox mice with transgenic strains expressing Cre recombinase under the control of tissue-speci®c promoters results in Nf1 inactivation in speci®c tissues. Using this approach, Nf1 has been inactivated speci®cally in neurons, Schwann cells and astrocytes. Neuron-speci®c Nf1 inactivation resulted in mice that demonstrate neurotrophin-independent dorsal root ganglion neuron survival in vitro and an overall reduction in brain cortical thickness. Aberrant neuronal survival was also observed in vitro, associated with increased cellularity and astrogliosis throughout cortex, hippocampus and brainstem [34]. Schwann-cell-speci®c Nf1 conditional knockout mice develop prominent Schwann-cell hyperplasia but few tumors [35]. Schwann-cell-speci®c Nf1 knockout mice generated to express one mutant and one wild-type allele in all cells (Nf1¯ox/mut; Krox20±Cre mice) develop plexiform neuro®broma-like tumors along peripheral nerves. Examination of the neuro®bromas in these mice demonstrates in®ltration of Nf1/ mast cells, suggesting that Nf1/ cells in the tumor environment may promote tumorigenesis. These ®ndings concur with previous mouse modeling experiments in which chimeric animals were generated by injecting Nf1 / embryonic stem cells into blastocyst-stage embryos. Only those embryos containing a small minority of Nf1 / cells survived to adulthood. Most of these mice developed multiple neuro®bromas reminiscent of plexiform neuro®bromas, suggesting that other neuro®bromin-de®cient cell types (e.g. mast cells) contributed to the development of these tumors [36].
In addition to growth-regulation defects, Nf1/ mice exhibit speci®c de®cits in learning and spatial memory, which likely re¯ects increased RAS activity and impairment of long-term potentiation. These Nf1/ learning abnormalities can be rescued by either genetic or pharmacologic manipulations of RAS activity or GABA receptor antagonists [33].
Astrocyte-speci®c Nf1 conditional knockout mice exhibit increased astrocyte proliferation both in vitro and in vivo, but do not develop gliomas even after 20 months of age [37]. In these studies, Nf1 inactivation in astrocytes by embryonic day 14 is not suf®cient for astrocytoma formation. These observations raise the intriguing possibility that either the cell of origin of NF1-associated gliomas is not the conventional glial ®brillary acidic protein-expressing astrocyte or that additional genetic changes are required.
Conditional Nf1 knockout mice
Cooperating genetic changes
The early embryonic lethal phenotype of the Nf1 knockout precludes a detailed analysis of the contribution of Nf1 to neuro®broma and astrocytoma formation. For this reason, Parada and co-workers have developed conditional knockout mice to facilitate the study of speci®c tissues lacking neuro®bromin expression [34]. Conditional Nf1 knockout mice result from tissue-speci®c bacCurrent Opinion in Genetics & Development 2003, 13:20±27
Another approach to studying NF1 tumor formation involves the generation of mice heterozygous for mutations in both Nf1 and other relevant tumor suppressor genes. On the basis of genetic studies of NF1-associated MPNSTs implicating the p53 tumor suppressor, investigators have generated Nf1/ ; p53/ mice [38]. These double heterozygote mice developed high-grade sarcowww.current-opinion.com
Neurofibromatosis 1 Dasgupta and Gutmann 25
mas with histopathologic features of human MPNST [38], demonstrating that loss of both neuro®bromin and p53 cooperate to facilitate MPNST formation. In a related study [39], 60% of p19ARF null mice generated on an Nf1/ background developed multiple tumors (sarcomas, lymphomas, leukemias and carcinomas), suggesting genetic cooperativity between these two tumor suppressor proteins in tumorigenesis. In addition, there are likely modifying genes that specify tumor susceptibility as well as the spectrum of speci®c tumors, as demonstrated using mice harboring both Nf1 and p53 mutations [40]. In this elegant study, Nf1/ ; p53/ mice developed highly aggressive glioblastoma multiforme brain tumors when maintained on the C57BL/6, compared to the SJL/J, genetic background. Aberrant EGFR signaling has also been observed in NF1-associated MPNSTs. Although EGFR is not normally expressed in Schwann cells, EGFR overexpression and signaling has been detected in MPNSTs from NF1 patients as well as transformed Nf1 / Schwann cells [19]. In addition, cell lines derived from mice heterozygous for mutations in both Nf1 and p53 overexpress EGFR and activate the PI3K-AKT pathway in response to EGFR stimulation [41].
Exon-speci®c Nf1 knockout mice
The existence of multiple neuro®bromin isoforms has raised the interesting possibility that speci®c isoforms have unique functions beyond RAS regulation. In an effort to address this hypothesis, Silva and co-workers have generated mice lacking exon 23a (involving the NF1±GAP-related domain [NF1-GRD]) and the brainspeci®c exon 9a (Y Elgersma, A Silva, personal communication). Targeted disruption of exon 23a resulted in learning impairments in mice, similar to conventional Nf1/ mice [42]. It is possible that the exon 23a knockout mice develop learning disabilities, not as a result of impaired RAS-GAP activity but rather because of reduced neuro®bromin activity attributable to exon 23a function, such as cAMP regulation. Further studies on exon 23a knockout mice may provide insights into the relationship between neuro®bromin and learning. The fact that these mice did not develop tumors likewise suggests that other functions of neuro®bromin attributable to the NF1±GRD may be important for growth regulation.
Conclusions
Signi®cant insights into the molecular pathogenesis of NF1-associated tumors have derived from studies of both human tumors and Nf1 knockout mice. The complementary information that results from these different approaches has raised interesting issues regarding the genetic and biological factors that contribute to tumorigenesis in individuals with NF1. It is clear that NF1 inactivation is suf®cient for the formation of some tumors www.current-opinion.com
in NF1, such as pheochromocytomas and leukemias, but is not suf®cient by itself to result in neuro®bromas, astrocytomas or MPNSTs. Whereas neuro®bromas require other participating Nf1/ cells to provide the proper cellular or mitogenic environment for tumor formation, astrocytoma formation may require Nf1 inactivation in speci®c astroglial progenitor cell populations or the presence of additional cooperating genetic changes. The notion that Nf1/ cells are not the biological equivalent of wild-type cells expands our appreciation of the role of tumor suppressor gene heterozygosity in growth control and tumorigenesis. It is highly likely that some of the clinical features of NF1 result solely from NF1 heterozygosity. As we enter into an era of targeted cancer therapeutics, an improved understanding of the signaling pathways dysregulated as a consequence of neuro®bromin loss will provide additional molecular targets for drug design. The future of clinical care for individuals with NF1 could one day involve drugs with highly selective activity towards speci®c biochemical pathways deranged in different cell types responsible for particular clinical features of NF1. Further work on the precise role of neuro®bromin in RAS and cAMP pathway regulation in speci®c cell types in both model organisms and human tissues may provide these essential insights.
References and recommended reading
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25. Kluwe L, Hagel C, Tatagiba M, Thomas S, Stavrou D, Ostertag H, von Deimling A, Mautner VF: Loss of NF1 alleles distinguish sporadic from NF1-associated pilocytic astrocytomas. J Neuropathol Exp Neurol 2001, 60:917-920. 26. Li J, Perry A, James CD, Gutmann DH: Cancer-related gene expression pro®les in NF1-associated pilocytic astrocytomas. Neurology 2001, 56:885-890. 27. Gutmann DH, Hedrick NM, Li J, Nagarajan R, Perry A, Watson MA: Comparative gene expression pro®le analysis of Neuro®bromatosis1-associated and sporadic pilocytic astrocytomas. Cancer Res 2002, 62:2085-2091. 28. Lakkis MM, Golden JA, O'Shea S, Epstein JA: Neuro®bromin de®ciency in mice causes excencephaly and is a modi®er for Splotch neural tube defects. Dev Biol 1999, 212:80-92. 29. Zhang Y, Vik TA, Ryder JW, Srour EF, Jacks T, Shannon K, Clapp DW: Nf1 regulates hematopoietic progenitor cell growth and Ras signalling in response to multiple cytokines. J Exp Med 1998, 187:1893-1902. 30. Gutmann DH, Loehr A, Zhang Y, Kim J, Henkemeyer M, Cashes A: Haploinsuf®ciency for neuro®bromatosis 1 (NF1) tumor suppressor results in increased astrocyte proliferation. Oncogene 1999, 18:4450-4459. 31. Gutmann DH, Wu YL, Hedrick NM, Zhu Y, Guha A, Parada LF: Heterozygosity for neuro®bromatosis 1 (NF1) tumor suppressor results in abnormalities in cell attachment, spreading and motility in astrocytes. Hum Mol Genet 2001, 10:3009-30016. These two reports [30,31] describe a direct effect of Nf1 heterozygosity on astrocyte proliferation and actin cytoskeleton-mediated processes. In these studies, the authors demonstrate that Nf1/ astrocytes exhibit increased cell proliferation, alterations in actin cytoskeleton-mediated processes, and dysregulated RAS activity. These observations support the notion that Nf1/ cells are not functionally equivalent to wild-type cells and may contribute to the pathogenesis of NF1-associated features. 32. Ingram DA, Hiatt K, King AJ, Fisher L, Shivakumar R, Christina D, Wenning MJ, Bruce D, Travers JB, Hood A et al.: Hyperactivation of p21ras and hematopoietic-speci®c Rho GTPase, Rac2, cooperate to alter the proliferation of neuro®bromin-de®cient mast cells in vivo and in vitro. J Exp Med 2001, 194:57-69. 33. Costa RM, Federov NB, Kogan JH, Murphu GG, Stern J, Ohno M, Kucherlapati R, Jacks T, Silva A: Mechanism for the learning de®cits in a mouse model of neuro®bromatosis type 1. Nature 2002, 415:526-530. 34. Zhu Y, Romero MI, Ghosh P, Ye Z, Charnay P, Rushing EJ, Marsh JD, Parada LF: Ablation of NF1 functions in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Genes Dev 2001, 15:859-876. 35. Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF: Neuro®bromas in NF1: Schwann cell origin and role of tumor environment. Science 2002, 296:920-922. This report describes the requirement for contributing Nf1/ cells to plexiform neuro®broma formation in mice lacking neuro®bromin in Schwann cells. This landmark study demonstrates for the ®rst time that neuro®bromin loss in Schwann cells by itself may not be suf®cient for tumorigenesis and that plexiform neuro®broma formation requires the contribution of Nf1/ cells (e.g. mast cells). 36. Cichowski K, Shih TS, Schmitt E, Santiago S, Reilly K, McLaughlin ME, Roderick TB, Bronson RT, Jacks T: Mouse models of tumor development in neuro®bromatosis type 1. Science 1999, 286:2172-2176. 37. Bajenaru ML, Zhu Y, Hedrick NM, Donahoe J, Parada LF, Gutmann DH: Astrocyte-speci®c inactivation of the neuro®bromatosis 1 gene (NF1) is insuf®cient for astrocytoma formation. Mol Cell Biol 2002, 22:5100-5113. This report demonstrates that neuro®bromin loss in maturing astrocytes is not suf®cient for astrocytoma formation in the mouse. In this study, the authors employ multiple approaches to inactivating neuro®bromin in astrocytes and ®nd that Nf1 loss in type-1 astrocytes does not confer a neoplastic phenotype. These ®ndings suggest that NF1-associated glioma formation may require additional cellular or genetic changes. 38. Vogel KS, Kleese LJ, Velasco-Miguel S, Meyers K, Rushing EJ, Parada LF: Mouse tumor model of neuro®bromatosis type 1. Science 1999, 286:2176-2179. www.current-opinion.com
Neurofibromatosis 1 Dasgupta and Gutmann 27
39. King D, Yang G, Thompson MA, Hiebert SW: Loss of neuro®bromatosis-1 and p19ARF cooperate to induce multiple tumor phenotype. Oncogene 2002, 21:4978-4982. 40. Reilly KM, Loisel DA, Bronson RT, McLaughlin ME, Jacks T: Nf1; Trp53 mice develop glioblastoma with evidence of strain-speci®c effects. Nat Genet 2000, 26:109-113. This report demonstrates that the genetic background has a signi®cant effect on tumor formation as well as the types of tumors generated in mice. In this study, the authors report that Nf1/ ; p53/ mice maintained on speci®cgenetic backgrounds develop brain tumorswhereasinotherstrains, brain tumor development is uncommon. These ®ndings suggest that additional `modi®er' genes exist that in¯uence tumorigenesis in the mouse. 41. Li H, Velasco-Miguel V, Vass CW, Parada LF, DeClue JE: Epidermal growth factor receptor signaling pathways are associated with tumorigenesis in the Nf1:p53 mouse tumor model. Cancer Res 2002, 62:4507-4513.
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This report describes the cooperative effect of EGFR expression and activation in Schwann cell tumor proliferation in mice developing MPNST. The authors ®nd that EGFR overexpression is seen in both mouse and human MPNSTs, suggesting that additional signaling abnormalities conferred by the aberrant expression of EGFR impact on the pathogenesis of one of the malignant tumor types in NF1. 42. Costa RM, Yang T, Huynh DP, Pulst SM, Viskochil DH, Silva AJ, Brannan CI: Learning de®cits, but normal development and tumor predisposition, in mice lacking exon 23a of Nf1. Nat Genet 2001, 27:399-405. This report describes the differential effects of exon 23a deletion on mouse development, tumor formation, and learning. In this study, the authors generate mice lacking exon 23a and demonstrate that these mice exhibit learning de®cits similar to Nf1/ mice, but are not prone to tumors. These ®ndings suggest that neuro®bromin-containing exon 23a contributes to learning and memory in mice, but has a limited role in tumor formation.
Current Opinion in Genetics & Development 2003, 13:20±27