NF1 Gene Expression Research Articles

Abstract 373: Silver nanoparticles are selectively cytotoxic to neurofibromin 1 deficient peripheral nerve sheath tumors

Abstract Neurofibromatosis type 1 (NF1) is the most common neurogenic disorder affecting 1 in every 3,000 people worldwide. NF1 is defined by heterozygous loss-of-function of the neurofibromin 1 gene (NF1). NF1 patients develop neurofibromas with near complete penetrance. These neurofibromas include peripheral nerve sheath tumors (PNSTs) such as plexiform neurofibromas (pNFs) and malignant PNSTs (MPNSTs). Current standard of care shows incomplete penetrance limiting clinical utility. Therefore, treatments to selectively and safely remove PNST cells would be advantageous. Biallelic loss of functional NF1 is the major driving force in the development of PNSTs. NF1 is a tumor suppressor gene responsible for suppressing Ras activity. Loss of NF1 increases Ras activity and subsequently increases intracellular reactive oxygen species (ROS). We and other groups have demonstrated that systemically administrable silver nanoparticles (AgNPs) are cytotoxic to a variety of cancers. We further showed that AgNP-mediated cytotoxicity is dependent upon intracellular ROS which induces rapid ionization (activation) of Ag0 to cytotoxic Ag+. Therefore, AgNPs represent a rational cancer-selective therapy for NF1-deficient PNSTs as these cells are enriched for ROS relative to non-cancerous cells. In this study, we found that AgNPs are significantly (~5-fold) more cytotoxic to NF1-null MPNST cells relative to tumor cell of origin Schwann cells and ~7-fold more cytotoxic to pNF cells relative to patient matched Schwann cells. Sporadic NF1 wild-type MPNST cells were not sensitive to AgNP. We further explored the relationship between oxidative stress and AgNP-mediated cytotoxicity in PNSTs. We found that co-administration of oxidative stress inducing cumene hydroperoxide with AgNP augments the cytotoxicity of AgNP in otherwise AgNP-tolerant cells (Schwann cells, NF1-wildtype MPNSTs). Importantly, either agent alone only showed modest cytotoxicity. As NF1 gene expression levels correlated with AgNP-sensitivity, we further studied this possible interaction by reintroducing NF1 into NF1-null MPNST. Remarkably, NF1 restoration decreased sensitivity to AgNP. This change is unique to AgNP as there was no altered sensitivity to standard of care chemotherapy doxorubicin. Conversely, knockdown of NF1 expression in Schwann cells increased sensitivity to AgNP with no corresponding change in sensitivity to pNF standard of care MEK inhibitor selumetinib. In conclusion, our work provides evidence that a therapeutic window exists for the safe use of AgNPs as a precision medicine to treat NF1-null PNSTs. Citation Format: Garrett Alewine, Adithya Ghantae, Christina Mamrega, Jerrica L. Knight, Bashnona Attiah, Robert A. Coover, Cale D. Fahrenholtz. Silver nanoparticles are selectively cytotoxic to neurofibromin 1 deficient peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 373.

Evaluation of NF1 and RASA1 gene expression in endometriosis.

BackgroundEndometrios affecting 6–10% of women of reproductive ages around the globe. Important pathways, including the MAPK and PI3K / Akt pathways, have been identified in the disease. The NF1 and RASA1 genes inactivate Ras by their own GTPase activity and controlled the high activity of these pathways.ObjectiveIn this study, we measured NF1 and RASA1 gene expression in the endometrial tissues of patients (eutopic and ectopic tissues) compared to the control samples.Materials and methodsIn our study, tissue samples were collected from 15 patients with endometriosis and 15 healthy women. We used quantitative polymerase chain reaction (qRT-PCR) to measure the NF1 and RASA1 gene expression levels in these samples.ResultsWe observed a significant decrease in the expression level of the NF1 gene in both eutopic and ectopic samples of endometriosis patients compared to control samples, while the expression of the RASA1 gene was significantly reduced only in ectopic tissues.

The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture.

Bone fracture is one of the most common injuries. Despite the high regenerative capacity of bones, failure of healing still occurs to near 10% of the patients. Herein, we aim to investigate the modulatory role of neurofibromatosis type I gene (NF1) to osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs) and new bone formation after fracture in a rat model. We studied the NF1 gene expression in normal and non‐union bone fracture models. Then, we evaluated how NF1 overexpression modulated osteogenic differentiation of BMSCs, autophagy activity, mTORC1 signalling and osteoclastic bone resorption by qRT‐PCR, Western blot and immunostaining assays. Finally, we injected lentivirus‐NF1 (Lv‐NF1) to rat non‐union bone fracture model and analysed the bone formation process. The NF1 gene expression was significantly down‐regulated in non‐union bone fracture group, indicating NF1 is critical in bone healing process. In the NF1 overexpressing BMSCs, autophagy activity and osteogenic differentiation were significantly enhanced. Meanwhile, the NF1 overexpression inhibited mTORC1 signalling and osteoclastic bone resorption. In rat non‐union bone fracture model, the NF1 overexpression significantly promoted bone formation during fracture healing. In summary, we proved the NF1 gene is critical in non‐union bone healing, and NF1 overexpression promoted new bone formation after fracture by enhancing autophagy and inhibiting mTORC1 signalling. Our results may provide a novel therapeutic clue of promoting bone fracture healing.

Experimental therapy for neurofibromatosis I shows promise

Experimental therapy for neurofibromatosis I shows promise

Abstract 1530: The obligate role of microglia in tumorigenesis in a mouse model of neurofibromatosis-1 optic glioma

Abstract Brain tumors (gliomas) are composed of heterogeneous populations of neoplastic and non-neoplastic cells. While the neoplastic cells have classically been the target of therapeutic drug design, recent studies have demonstrated a critical role for non-neoplastic cells in brain tumor growth. The importance of non-neoplastic (stromal) cells is underscored by genetically-engineered mouse (GEM) models of Neurofibromatosis type 1 (NF1)-associated optic glioma. Whereas mice lacking Nf1 gene expression in GFAP+ glial cells do not develop brain tumors, nearly 100% of Nf1+/− mice with GFAP+ cell Nf1 inactivation form optic gliomas. These observations establish an obligate role for Nf1+/− non-neoplastic cells in gliomagenesis. One of the non-neoplastic cell types found in both mouse and human NF1-associated gliomas is microglia, a resident population of macrophage-like cells. To prove that microglia are essential drivers of glioma formation, we generated Nf1 optic glioma mice with reduced expression of the microglial chemokine receptor CX3CR1 (Nf1+/−GFAPCKO-cx3cr1+/GFP mice). Consistent with the critical role of CX3CR1 in microglial function, Nf1+/−GFAPCKO-cx3cr1+/GFP mice had reduced numbers of Iba1+ microglia in the optic nerve. Moreover, Nf1+/−GFAPCKO-cx3cr1+/GFP mice had optic glioma proliferative indices and optic nerve volumes comparable to those found in wild-type mice, demonstrating that microglia are necessary for optic nerve gliomagenesis. Collectively, these findings establish an obligate role for microglia in glioma formation and suggest that CX3CR1 is a potential target for future therapeutic drug design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1530. doi:1538-7445.AM2012-1530

The neurofibromin 1 type I isoform predominance characterises female population affected by sporadic breast cancer: preliminary data

AimsNeurofibromin 1 (NF1) as a tumour suppressor gene can give rise to several transcripts by an alternative splicing event, generated at least for CELF cofactors. At present, the NF1 isoforms...

Long-range DNA interactions are specifically altered by locked nucleic acid-targeting of a CTCF binding site

Long-range DNA interactions play an important role in gene expression. CCCTC-binding factor (CTCF), a ubiquitously expressed and evolutionarily conserved 11-zinc-finger DNA binding protein, is intimately involved in gene regulation, helping to establish and maintain chromatin architecture and long-range DNA interactions. In order to study the effects of manipulating long range chromatin interactions in the regulation of the neurofibromatosis gene NF1, we targeted Zorro locked nucleic acids (Zorro LNA) to a single CTCF binding site at an NF1 locus in human fibroblast cells. Using chromatin immunoprecipitation, we determined that this Zorro LNA altered CTCF and RNA polymerase II binding at three separate and distinct regions in the NF1 gene. This change in protein binding was associated with changes in long-range DNA interactions at the NF1 locus and downregulation of NF1 gene expression. This study describes an efficient and convenient method to manipulate chromatin structure and alter gene expression that is regulated by long-range DNA interactions without changing the DNA sequence. The use of specific Zorro LNA probes may facilitate our efforts to understand the interactions between chromatin architecture and gene expression.

Deletion of the Tumor Suppressor Gene NF1 Is Found In 3.5% of 485 De Novo Adult Myeloid Leukemia and Is Correlated with Unfavourable Cytogenetic: On Behalf of the ALFA Group

AbstractAbstract 4171NF1 acts as a tumor-suppressor gene by encoding neurofibromin1, a GTPase-activating protein (GAP) inhibiting Ras signaling pathway. Germline mutations or microdeletions of NF1 are responsible for neurofibromatosis type 1, and the somatic loss of the remained wild-type allele lead to malignant tumors or juvenile myelomonocytic leukemia (JMML). Furthermore, several studies revealed heterozygous somatic deletions of the 17q11.2 region including NF1 in adult myeloid malignancies. The reported frequencies of this abnormality varied between 2.6% and 11% in AML and this variation can be attributable to heterogeneity or size of the analysed cohorts.Previously, we analyzed 131 de novo AML cases (AML3 excluded) by Agilent™ 105K microarrays. 6/131 cases (4.6%) showed somatic deletions in 17q11.2, including a small minimal deleted region of 300 kb comprising the entire NF1 gene. To further investigate the incidence of NF1 deletion in de novo AML, 354 additional patients were therefore screened for the deletion by quantitative real-time PCR (Primers and TaqMan-based probe Hs 01778367_cn from Applied Biosystems), and FISH (NF1/MPO probe KBI-40144 from Kreatech) was performed to confirm the loss of NF1 copy number. Altogether, heterozygous NF1 deletion was observed in 17/485 (3.5%) de novo AML. Clinico-biological data were available from 14 NF1 deleted patients and 380 non-deleted patients included in the ALFA-9801 and 9802 French Trials. There were no significant differences between the 2 groups in age, sex ratio, leukocytosis, FAB classification of AML, mutational status of FLT3, NPM1, CEBPα and IDH. Interestingly, NF1 deletion was significantly correlated with unfavourable cytogenetic (50% vs 18%, p=0.008) and especially with monosomal karyotype (29% vs 9%, p=0.03). However, no statistical significant differences were observed for complete remission rate, relapse risk 3 years after diagnosis and 3-years overall survival. Screening for bi-allelic inactivation by sequencing the remained allele in NF1 deleted patients is in progress.We next evaluated NF1 gene expression for 93 patients of our cohort (3 with NF1 deletion and 90 without) by Affymetrix U133 Plus 2.0 microarrays. The 3 NF1 deleted patients revealed a significant reduced mean of NF1 expression level. Interestingly, about 10% of the NF1 non-deleted patients presented a similar decrease in NF1 expression rate. This suggests that mechanisms for transcriptional regulation (such as mutations or epigenetic silencing of NF1) may also contribute to AML pathogenesis.In conclusion, NF1 deletions occur in only 3.5% of de novo AML and are associated with unfavourable cytogenetic. This relatively low frequency of NF1 deletion can however be counterbalanced by others alterations acting at the transcriptional level and this remains to be investigated. Disclosures:No relevant conflicts of interest to declare.

Protective effect of the dopamine D3 receptor agonist (7-OH-PIPAT) against apoptosis in malignant peripheral nerve sheath tumor (MPNST) cells

Emerging evidence indicates that the dopamine D(3) receptor (D(3)R) mediates protective roles both in neuronal and non-neuronal cell lines. In a previous study we proposed that neurofibromin, a large tumor suppressor protein encoded by the neurofibromatosis type 1 gene (NF1), may increase susceptibility to apoptosis after serum deprivation in malignant peripheral nerve sheath tumor (MPNST) cells, thus acting as a proapoptotic gene. In addition, it has been observed that D(3)Rs are functionally correlated to neurofibromin. In this study, we examined whether 7-OH-PIPAT, a potent dopamine D(3)R agonist, exerts an antiapoptotic role under the same culture conditions and then correlated this effect to changes in NF1 expression. Results showed that serum deprivation caused a significant reduction of cell viability (MTT assay) both after 24 and 48 h (p<0.001). Treatment with increasing concentrations of 7-OH-PIPAT (10(-9)-10(-5) M) induced a progressive increase in cell viability both after 24 and 48 h as compared to vehicle-treated cells, with significant changes at the highest concentrations tested (10(-6) and 10(-5) M). Consistently, at the latter two concentrations, a significant reduction in oligonucleosomes formation was observed, thus suggesting an antiapoptotic role of 7-OH-PIPAT. These results were confirmed by Hoechst 33254 nuclear staining. To investigate whether these effects were correlated to changes in NF1 transcript and protein expression, quantitative real-time PCR, Western blot and immunofluorescence analyses were performed. Results demonstrated that the upregulation of NF1 transcripts and protein levels induced by serum withdrawal were remarkably attenuated by 10(-6) and 10(-5) M agonist treatment within 24 h (p<0.01 and p<0.001, respectively), whereas similar effects were observed already at a lower concentration (10(-7) M) after 48 h treatment (p<0.001). In conclusion, these results suggest that D(3)R might mediate the protective response to serum deprivation in MPNST cells through the inhibition of NF1 gene expression, further underlying a subtle role of these receptors in MPNST development.

Deletion of the Tumor Suppressor Gene NF1 Is An Alternative Mechanism for Aberrant Activation of the RAS Pathway and Is Found in 11% of Acute Myeloid Leukemia.

Abstract 401The NF1 gene encodes neurofibromin 1, a Ras-specific guanosine triphosphatase-activating protein (GAP) that negatively regulates the p21ras (Ras) family of signaling proteins by accelerating GTP hydrolysis. Germline loss of function mutations of NF1 lead to Neurofibromatosis type I. Carriers of the mutations develop benign neurofibromas and are predisposed to neuronal tumors, but also to juvenile myelomonocytic leukemia (JMML), MDS, and AML. In addition, in children suffering from CMML without germline NF1 mutations, acquired loss of function mutations of NF1 have been reported. In contrast, the role of NF1 in adult myeloid malignancies has not been studied in detail. Therefore, we first evaluated NF1 gene expression in 272 AML cases which were analyzed by Affymetrix HG-U133 Plus 2.0 microarrays. These included cases with t(15;17)(q22;q12) (n=15), t(8;21)(q22;q22) (n=16), inv(16)(p13q22) (n=7), t(11q23)/MLL-rearrangement (n=10), complex aberrant karyotype (n=47), normal karyotype (n=97), and with various other genetic abnormalities (n=80). The median NF1 expression intensity was 131.6 (range 35.2 - 457.5). 68 cases showed an expression intensity of NF1 below 98.6 (first quartile). In this cohort cases with t(8;21) (n=10) or complex karyotype (n=18) were over-represented (Chi-square: p<0.0001 and p=0.021, respectively), while cases with normal karyotype (n=16) were under-represented (Chi-square: p=0.016). In 54/68 of the latter cases with low expression material was availabel for FISH analysis with a probe spanning the NF1 locus. Remarkably, in 11/54 of these cases (20.4%) a NF1 deletion was observed by interphase FISH (% of cells with NF1 deletion median 90% (range: 60-99%). The mean±SD NF1 expression intensity in cases with NF1 deletion was 60.4±17.7 as compared to 75.5±18.0 in cases with 2 NF1 copies (p=0.023). Chromosome banding analysis in these 11 cases revealed a complex karyotype (n=7), a normal karyotype (n=2), an inv(3)(q21q26), and a 5q-deletion accompanied by trisomy 21, respectively. To further investigate the incidence of NF1 deletion in myeloid malignancies 425 additional patients were analyzed by FISH for NF1 deletion using a 420 kb probe spanning the NF1 gene. A heterozygous NF1 deletion was observed in 30/425 (7.1%) patients: de novo AML: 13/142 (9.2%), s-AML: 5/39 (12.8%), t-AML: 4/13 (31%), CMML: 5/99 (5.6%), MDS: 0/122 (0%), MPN: 3/10 (30%). Chromosome banding analysis in the NF1-deleted cases revealed a normal karyotype (n=4), an inv(16)(p13q22) (n=6), an inv(3)(q21q26) (n=6), a complex aberrant karyotype (n=5) or other abnormalities (n=9). In conclusion, NF1 deletions occur in 11% of AML, 5% of CMML and 3/10 MPN and therefore are a frequent and important alternative genetic mechanism for activating the RAS pathway in adult myeloid malignancies. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Nf1 expression is dependent on strain background: implications for tumor suppressor haploinsufficiency studies

Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with elevated risk for both astrocytoma and peripheral nerve sheath tumors. NF1 is caused by a germline mutation in the NF1 gene, with tumors showing loss of the wild type copy of NF1. In addition, NF1 heterozygosity in surrounding stroma is important for tumor formation, suggesting an additional role of haploinsufficiency for NF1. Studies in mouse models and NF1 families have implicated modifier genes unlinked to NF1 in the severity of the disease and in susceptibility to astrocytoma and peripheral nerve sheath tumors. To determine if differences in Nf1 expression may contribute to the strain-specific effects on tumor predisposition, we examined the levels of Nf1 gene expression in mouse strains with differences in tumor susceptibility using quantitative polymerase chain reaction. The data presented in this paper demonstrate that strain background has as much effect on Nf1 expression levels as mutation of one Nf1 allele, indicating that studies of haploinsufficiency must be carefully interpreted with respect to strain background. Because expression levels do not correlate entirely with the susceptibility or resistance to tumors observed in the strain, these data suggest that either variation in Nf1 levels is not responsible for the differences in astrocytoma and peripheral nerve sheath tumor susceptibility in Nf1-/+;Trp53-/+cis mice, or that certain mouse strains have evolved compensatory mechanisms for differences in Nf1 expression.

NF1 tumor suppressor in epidermal wound healing with special focus on wound healing in patients with type 1 neurofibromatosis

Type 1 neurofibromatosis syndrome (NF1) has been linked with mutations of the NF1 gene which encodes tumor suppressor neurofibromin, a regulator of Ras-MAPK signaling. In human epidermis, keratinocytes express NF1 tumor suppressor and it may have a distinctive function in these cells during wound healing, such as regulating Ras activity. NF1 expression was first studied during the epidermal wound healing using suction blister method. NF1 gene expression increased both in hypertrophic and migrating zones of the healing epidermis, and also in dermal fibroblasts underneath the injury. This prompted us to study epidermal wound healing in NF1 patients. Wound healing efficiency was evaluated 4 days after blister induction by clinical, physiological and histological methods. Epidermal wound healing was equally effective in NF1 patients and healthy controls. In addition, dermal wound healing appears to function normally in NF1 patients based on retrospective and follow-up study of biopsy scars. Furthermore, the healing wounds were analyzed immunohistochemically for cell proliferation rate and Ras-MAPK activity. Neither epidermal keratinocytes nor dermal fibroblasts showed difference in the cell proliferation rate or Ras-MAPK activity between NF1 patients and controls. Interestingly, NF1 patients displayed increased cell proliferation rate and Ras-MAPK activity in periarteriolar tissue underneath the wound. The results of the study suggest that epidermal wound healing is not markedly altered in NF1 patients. Furthermore, NF1 protein seems not to have an important function as a Ras-MAPK regulator in epidermal keratinocytes or dermal fibroblasts but instead appears to be regulator of Ras-MAPK signaling in vascular tissues.

NF1 tumor suppressor protein and mRNA in skeletal tissues of developing and adult normal mouse and NF1-deficient embryos.

NF1 is a heritable disease with multiple osseous lesions. The expression of the NF1 gene was studied in embryonic and adult rodent skeleton and in NF1-deficient embryos. The NF1 gene was expressed intensely in the cartilage and the periosteum. Impaired NF1 expression may lead to inappropriate development and dynamics of bones and ultimately to the osseous manifestations of the disease. Neurofibromatosis type 1 is caused by mutations in the NF1 gene encoding the Ras GTPase activating protein (Ras-GAP) neurofibromin. Skeletal ailments such as short stature, kyphoscoliosis, and tibial bowing and pseudarthrosis are common osseous manifestations of NF1. These symptoms are congenital, implying a role for neurofibromin in proper bone growth. However, little is known about its expression in skeletal tissues during their development. The expression of the NF1 gene was studied in normal and NF1+/- mouse fetuses at embryonic days 12.5-15.5 and in skeletal tissues of adult mice and rats. In situ hybridization, immunohistochemistry, and Western blot analysis were used to identify the NF1 gene expression profile. NF1 mRNA and protein were elevated in resting, maturation, and hypertrophic chondrocytes at the growth plate. Parallel studies on NF1+/- embryos showed expression patterns identical to wildtype. The periosteum, including osteoblasts and osteoclasts, and osteocytes of the cortical bone of adult mice were also intensely labeled for NF1 protein and mRNA. Western transfer analysis detected NF1 protein in the respective rat tissues. Phosphorylation of p42 and p44 MAP kinases, the downstream consequence of Ras activation, was elevated in hypertrophic chondrocytes of NF1+/- embryos. The results suggest that neurofibromin may act as a Ras-GAP in skeletal cells to attenuate Ras transduced growth signals and thus play a role during ossification and dynamics of bone. Loss of NF1 function may therefore lead to dysplastic bone growth, thereby causing the debilitating osseous symptoms of NF1.

Mlh1 deficiency accelerates myeloid leukemogenesis in neurofibromatosis 1 (Nf1) heterozygous mice.

Defects in DNA mismatch repair (MMR) have been implicated in the genesis of a diverse set of human cancers. Recent studies have suggested that one of the targets of MMR is the neurofibromatosis 1 (NF1) gene. To evaluate the contribution of Mlh1 MMR deficiency to Nf1 tumorigenesis, Mlh1-/-;Nf1+/- mice were generated. All Mlh1-/-;Nf1+/- mice (n=21) were dead by 260 days compared to none of the Nf1+/- mice. In all, 50% of the Mlh1-/-;Nf1+/- mice were dead at 150 days compared to 252 days for Mlh1-/- mice. Nine of the Mlh1-/-;Nf1+/- mice were found to harbor intrathoracic NOS2-immunoreactive myeloid leukemias similar to the hematopoietic malignancies observed in older Nf1+/- mice. As expected, significant microsatellite instability was observed in six of six tumors and neurofibromin expression was lost in all tumors analysed. These results suggest that MMR deficiency can accelerate myeloid leukemogenesis in Nf1+/- mice, presumably by inactivating Nf1 gene expression.

Mutational and expression analysis of the NF1 gene argues against a role as tumor suppressor in sporadic pilocytic astrocytomas.

Children with neurofibromatosis type I (NF1) have a highly increased risk for developing optic nerve gliomas. Several lines of evidence support the notion that the NF1 gene functions as tumor suppressor in these pilocytic astrocytomas and therefore it is tempting to hypothesize that the NF1 gene plays a similar role in sporadic pilocytic astrocytomas. We searched for possible mechanisms of inactivation of the NF1 gene in pilocytic astrocytomas of different locations. Protein truncation testing (PTT) did not render indication for inactivating mutations in 10 analyzed tumors. Further, loss of heterozygosity analysis revealed maintenance of heterozygosity for 3 intragenic markers in 11 informative cases. Using a real-time PCR-based assay we showed that total NF1 transcript levels are high in pilocytic astrocytomas and that the NF1 type I and type II expression ratios in pilocytic astrocytomas are comparable to ratios in normal brain tissue and high-grade gliomas. Consequently, the data presented here argue against altered NF1 gene expression and the involvement of the NF1 gene in the tumorigenesis of sporadic pilocytic astrocytomas.

Heterozygosity for the neurofibromatosis 1 (NF1) tumor suppressor results in abnormalities in cell attachment, spreading and motility in astrocytes.

Individuals with the neurofibromatosis 1 (NF1) tumor predisposition syndrome develop low-grade pilocytic astrocytomas at an increased frequency. Previously, we demonstrated that astrocytes from mice heterozygous for a targeted mutation in the Nf1 gene (Nf1+/- astrocytes) exhibit a cell autonomous growth advantage associated with increased RAS pathway activation. In this report, we extend our initial characterization of the effect of reduced Nf1 gene expression on astrocyte function by demonstrating that Nf1+/- astrocytes exhibit decreased cell attachment, actin cytoskeletal abnormalities during the initial phases of cell spreading, and increased cell motility. Whereas these cytoskeletal abnormalities were also observed in Nf1-/- astrocytes, astrocytes expressing a constitutively active RAS molecule showed increased cell motility and abnormal actin cytoskeleton organization during cell spreading, but exhibited normal cell attachment. Based on ongoing gene expression profiling experiments on human astrocytoma tumors, we demonstrate increased expression of two proteins implicated in cell attachment, spreading and motility (GAP43 and T-cadherin) in Nf1+/- and Nf1-/- astrocytes. These results support the emerging notion that tumor suppressor gene heterozygosity results in abnormalities in cell function that may contribute to the pathogenesis of non-tumor phenotypes in NF1.

Neurofibromatosis type 1: II. Answers from animal models

Neurofibromatosis type 1 (NF1), or von Recklinghausen’s disease, is a complex disease resulting from mutations in the Nf1 gene. Moreover, the product of the NF1 gene neurofibromin also plays a very important role(s) in various aspects of the normal development and physiology of the organism. Although NF1 has been recognized for centuries, it is only relatively recently that there has been progress in the identification, isolation, and characterization of the NF1 gene and its protein product, neurofibromin (Seizinger et al., 1987; Fountain et al., 1989; Wallace et al., 1990; Marchuck et al., 1991). Nevertheless, progress in understanding the molecular regulation(s) and in identifying the molecular and cellular targets underlying the NF1 disease has lagged. There is much to be learned about the regulation(s) of the Nf1 gene expression and the function(s) and molecular interactions of neurofibromin. The role of neurofibromin in producing the multiple symptoms of the disease in the various cell types remains an enigma. Furthermore, there is still some controversy regarding the primary cellular target that in NF1 leads to neurofibroma and other NF1-associated symptoms and signs. This is the second part of a two-part review on NF1. The first part includes a general overview of the NF1 disease, the NF1 gene, and the genetic and cellular basis of the NF1 disease, with reference to controversial issues that hamper the full understanding of the disease and consequently hinder the efforts to find a cure (Lakkis and Tennekoon, 2000). In this part, the focus is on the animal models of NF1 disease, and we review both in vivo studies on these animals and in vitro studies using cells derived from the Nf12/2 mice. Although these studies have helped us to address some unanswered questions and enhance our knowledge about the molecular and cellular basis of NF1, additional questions have arisen that require further studies. Genetic manipulations in animals, especially in the mouse, have provided very good models to study and improve our understanding of human genetic diseases. The disruption of the Nf1 gene in the mouse is no exception. The targeted disruption of the Nf1 gene in the mouse (Jacks et al., 1994; Brannan et al., 1994) has not only provided new insights into the role of this gene in NF1 pathogenesis but has also revealed new and unexpected roles for NF1 gene in normal growth and development. Although the heterozygous mice appeared, in general, to be normal and did not display the classical symptoms of NF1 disease, Jacks et al. (1994) observed that, as these mice aged, they demonstrated a greater predisposition to a variety of tumor types, including lymphoma, lung adenocarcinoma, hepatoma, and fibrosarcoma. Furthermore, some of these mice developed tumors that are characteristically seen in human NF1, such as neurofibrosarcoma, pheochromocytoma, and myeloid leukemia. Cytological examination of tumor material showed that the wild-type Nf1 allele was lost in approximately half of the tumors, supporting the view that loss of heterozygosity (LOH) is required for tumorigenesis, thus making these mice a potential model for human disease. On the other hand, the homozygous mutant mice display a variety of developmental abnormalities. The most critical is a developmental cardiac defect that causes major circulatory dysfunction, leading to midgestation lethality (between days 12.5 and 14 of gestation). The mutant mice display generalized edema with systemic vascular congestion and signs of hemorrhage, all indicative of cardiac failure. Anatomical and histological analyses of the mutant hearts reveal many abnormalities, including double-outlet right ventricle (DORV) and ventricular septal defect (VSD) in addition to a dramatic enlargement of the endocardial cushions in the outflow tract in the atrioventricular region. The enlarged endocardial cushions most likely represent the critical abnormality, causing blockage of the inflow of circulating blood to the heart and causing the ultimate lethality. This endocardial cushion abnormality is caused by Ras up-regulation because of the absence of normal Nf1 gene product (Lakkis and Epstein, 1998), suggesting that this developmental process is regulated by

URINARY BLADDER TRANSITIONAL CELL CARCINOGENESIS IS ASSOCIATED WITH DOWN-REGULATION OF NF1 TUMOR SUPPRESSOR GENE IN VIVO AND IN VITRO

The NF1 gene product (neurofibromin) is known to act as a tumor suppressor protein by inactivating ras. The best documented factors involved in urinary bladder transitional cell carcinoma (TCC) are ras proto-oncogene activation and p53 suppressor gene mutations. This is the first study reporting alterations in NF1 gene expression in TCC. We examined NF1 gene expression in a total of 29 surgical urinary bladder TCC specimens representing grades 1 to 3 and in three cell lines, RT4, 5637, and T24 (representing grades 1 to 3, respectively). Decreased NF1 gene expression was observed in 23 of 29 (83%) TCC specimens as estimated by immunohistochemistry, the decrease being more pronounced in high-grade tumors. NF1 mRNA levels were markedly lower in TCC tissue compared with adjacent non-neoplastic urothelium, as studied by in situ hybridization for grade 3 TCC. Immunohistochemistry and Western blotting demonstrated that TCC cell lines expressed NF1 protein at different levels, expression being almost undetectable in T24 (grade 3) cells. Northern blotting for cell lines demonstrated reduced NF1 mRNA levels in grade 3 TCC cells. Reverse transcription polymerase chain reaction for cell lines and selected grade 2 and grade 3 tissue samples demonstrated NF1 type II mRNA isoform predominance in all samples studied. Our results show that both NF1 mRNA and protein levels are decreased in high-grade TCC, suggesting that alterations of NF1 gene expression may be involved in bladder TCC carcinogenesis.

Urinary Bladder Transitional Cell Carcinogenesis Is Associated with Down-Regulation of NF1 Tumor Suppressor Gene in Vivo and in Vitro

The NF1 gene product (neurofibromin) is known to act as a tumor suppressor protein by inactivating ras. The best documented factors involved in urinary bladder transitional cell carcinoma (TCC) are ras proto-oncogene activation and p53 suppressor gene mutations. This is the first study reporting alterations in NF1 gene expression in TCC. We examined NF1 gene expression in a total of 29 surgical urinary bladder TCC specimens representing grades 1 to 3 and in three cell lines, RT4, 5637, and T24 (representing grades 1 to 3, respectively). Decreased NF1 gene expression was observed in 23 of 29 (83%) TCC specimens as estimated by immunohistochemistry, the decrease being more pronounced in high-grade tumors. NF1 mRNA levels were markedly lower in TCC tissue compared with adjacent non-neoplastic urothelium, as studied by in situ hybridization for grade 3 TCC. Immunohistochemistry and Western blotting demonstrated that TCC cell lines expressed NF1 protein at different levels, expression being almost undetectable in T24 (grade 3) cells. Northern blotting for cell lines demonstrated reduced NF1 mRNA levels in grade 3 TCC cells. Reverse transcription polymerase chain reaction for cell lines and selected grade 2 and grade 3 tissue samples demonstrated NF1 type II mRNA isoform predominance in all samples studied. Our results show that both NF1 mRNA and protein levels are decreased in high-grade TCC, suggesting that alterations of NF1 gene expression may be involved in bladder TCC carcinogenesis.

DNA Sequences in the Promoter Region of the NF1 Gene Are Highly Conserved between Human and Mouse

The gene for type 1 neurofibromatosis (NF1) is most highly expressed in brain and spinal cord, although low levels of mRNA can be found in nearly all tissues. As a first step in investigating the regulation of NF1 gene expression, we have cloned and sequenced the promoter regions of the human and mouse NF1 genes and mapped the transcriptional start sites in both species. We report here that the 5' ends of the human and murine NF1 genes are highly conserved. While no discernable TATA or CCAAT box sequences are seen, transcription initiates at identical sites in both species, 484 nucleotides upstream of the ATG initiation codon in the human gene. The human and mouse NF1 genes share particularly high sequence homology (95%) between nucleotides -33 and +261 and contain several perfectly conserved transcription factor binding site motifs, including a cAMP response element, several AP2 consensus binding sites, and a serum response element. The high conservation of these sequences indicates that they are likely to be significant in the regulation of NF1 gene expression.