NF2 Inactivation Research Articles

The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma

Malignant pleural mesotheliomas (MPMs) often show CDKN2A and NF2 inactivation, but other highly recurrent mutations have not been described. To identify additional driver genes, we used an integrated genomic analysis of 53 MPM tumor samples to guide a focused sequencing effort that uncovered somatic inactivating mutations in BAP1 in 23% of MPMs. The BAP1 nuclear deubiquitinase is known to target histones (together with ASXL1 as a Polycomb repressor subunit) and the HCF1 transcriptional co-factor, and we show that BAP1 knockdown in MPM cell lines affects E2F and Polycomb target genes. These findings implicate transcriptional deregulation in the pathogenesis of MPM.

Identification of a progenitor cell of origin capable of generating diverse meningioma histological subtypes

Meningiomas are among the most common primary central nervous system tumours in adults. Studies focused on the molecular basis for meningioma development are hampered by a lack of information with regard to the cell of origin for these brain tumours. Herein, we identify a prostaglandin D synthase-positive meningeal precursor as the cell of origin for murine meningioma, and show that neurofibromatosis type 2 (Nf2) inactivation in prostaglandin D2 synthase (PGDS) (+) primordial meningeal cells, before the formation of the three meningeal layers, accounts for the heterogeneity of meningioma histological subtypes. Using a unique PGDSCre strain, we define a critical embryonic and early postnatal developmental window in which biallelic Nf2 inactivation in PGDS (+) progenitor cells results in meningioma formation. Moreover, we identify differentially expressed markers that characterize the two major histological meningioma subtypes both in human and mouse tumours. Collectively, these findings establish the cell of origin for these common brain tumours as well as a susceptible developmental period in which signature genetic mutations culminate in meningioma formation.

The Merlin/NF2 Tumor Suppressor Functions through the YAP Oncoprotein to Regulate Tissue Homeostasis in Mammals

The conserved Hippo signaling pathway regulates organ size in Drosophila and mammals. While a core kinase cascade leading from the protein kinase Hippo (Hpo) (Mst1 and Mst2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined, especially in mammals. Using conditional knockout mice, we demonstrate that the Merlin/NF2 tumor suppressor and the YAP oncoprotein function antagonistically to regulate liver development. While inactivation of Yap led to loss of hepatocytes and biliary epithelial cells, inactivation of Nf2 led to hepatocellular carcinoma and bile duct hamartoma. Strikingly, the Nf2-deficient phenotypes in multiple tissues were largely suppressed by heterozygous deletion of Yap, suggesting that YAP is a major effector of Merlin/NF2 in growth regulation. Our studies link Merlin/NF2 to mammalian Hippo signaling and implicate YAP activation as a mediator of pathologies relevant to Neurofibromatosis 2.

Effect of EGFR and IGF1R on feedback activation of AKT arising from inhibition of mTOR in malignant pleural mesothelioma (MPM).

e13607 Background: NF2 inactivation is one of the major genetic alterations in MPM and has recently been causally associated with activation of the mTOR pathway. In other cancers, inhibition of the...

Abstract 1128: The tumor suppressive function of Merlin/Nf2 is mediated by a novel tight junction-associated protein complex

Abstract Mutations in the NF2 tumor suppressor gene lead to inherited cancer-disorder Neurofibromatosis Type 2 (NF2) and other sporadic nerve-sheath tumors. Merlin, the protein product of the NF2 gene, suppresses cell proliferation and tumorigenesis by regulating multiple signaling pathways including the Ras-MAPK pathway. Through affinity purification, we have identified a novel tight-junction-associated protein complex comprising Merlin and a number of tight junction associated proteins. We show that Merlin directly interacts and co-localizes with one of these tight junction proteins through their mutual coiled-coil domains in epithelial and Schwann cells. Furthermore, the two proteins co-localize to paranodes and Schmidt-Lantermann incisures in myelinated peripheral nerve, the most frequent site of tumor development caused by Nf2 inactivation. We establish that the novel Merlin-binding protein functions downstream of Merlin as a positive regulator of Rac1 activity and the Ras-MAPK signaling. Importantly, we are able to demonstrate that depletion of this Merlin-interacting protein in Nf2-/- schwannoma cells down-regulates the Ras-MAPK signaling pathway, and impedes cellular proliferation in vitro and tumor development in vivo. Taken together with our observations that several NF2 patient-derived mutant Merlin proteins exhibit diminished association with this protein and that its level is elevated in 9 out of 10 human schwannoma samples examined, we conclude that the novel Merlin-binding partner identified in this study mediates the tumor suppressive function of Merlin and represents a potential therapeutic target for NF2 related tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1128.

Tumor-suppression functions of merlin are independent of its role as an organizer of the actin cytoskeleton in Schwann cells

Merlin is the product of the Nf2 tumor-suppressor gene, and inactivation of Nf2 leads to the development of neural tumors such as schwannomas and meningiomas in humans and mice. Merlin is a member of the ERM (ezrin, radixin and moesin) family of proteins that function as organizers of the actin cytoskeleton. Merlin structure is thought to be similar to that of the ERM proteins, and is held in a closed clamp conformation via intramolecular interactions of its N-terminal FERM (four-point-one, ERM) domain with an alpha-helical C-terminal domain. Like ERMs, merlin can remodel actin-rich cortical structures, yet merlin uniquely inhibits the proliferation of many different cell types. Here, we report that the F2 subdomain of the FERM domain and a domain close to the C-terminus that is defined by residues 532-579 are essential for merlin-mediated inhibition of primary Schwann cell proliferation. Furthermore, we demonstrate that the F1 subdomain of the merlin FERM domain is required for actin colocalization, proper regulation of merlin C-terminal phosphorylation and for remodeling the cytoskeleton, yet is not required for the inhibition of Schwann cell proliferation. Thus, tumor suppression by merlin is independent of its role as an organizer of the actin cytoskeleton in Schwann cells.

Proteolytic processing of protein-tyrosine phosphatase receptor type Z

Primary human cells can be transformed into tumor cells by a defined set of genetic alterations including telomerase, oncogenic RasV12, and the tumor suppressors p53 and pRb. SV40 small T (ST) is required for anchorage-independent growth in vitro and in vivo. Here, we identify the Hippo tumor suppressor pathway as a critical target of ST in cellular transformation. We report that ST uncouples YAP from the inhibitory activity of the Hippo pathway through PAK1-mediated inactivation of NF2. Membrane-tethered activated PAK is sufficient to bypass the requirement for ST in anchorage-independent growth. PAK acts via YAP to mediate the transforming effects of ST. Activation of endogenous YAP is required for ST-mediated transformation and is sufficient to bypass ST in anchorage-independent growth and xenograft tumor formation. Our findings uncover the Hippo tumor suppressor pathway as a final gatekeeper to transformation and tumorigenesis of primary cells.

Microsatellite Analysis of Recurrent Vestibular Schwannoma (Acoustic Neuroma) Following Stereotactic Radiosurgery

Genetic and immunohistochemical studies may provide insight into the mechanisms of vestibular schwannoma (VS) recurrence following radiation therapy. Stereotactic radiation therapy is an increasingly common alternative to microsurgical resection for the primary management of sporadic VS. The molecular mechanisms associated with recurrent vestibular schwannoma (VS) following radiation therapy are not known. Primary or irradiated VS tumors were fresh-frozen at the time of surgical resection and microdissected to undergo DNA extraction. Lymphocytic control DNA was isolated from blood obtained by venipuncture. Paired normal and tumor DNA specimens were analyzed for allelic loss by PCR amplification of polymorphic dinucleotide repeat sequences. Immunohistochemical studies were performed on paraffin-embedded, irradiated surgical specimens. Using 16 polymorphic microsatellite markers, 20 of 26 non-irradiated VS demonstrated loss of heterozygosity (LOH) in at least one locus of chromosome 22q. In contrast, none of the four irradiated recurrent VS demonstrated LOH on chromosome 22q (p = 0.008). No allelic loss was seen in either the primary or irradiated VS utilizing markers mapping to chromosome 10. Deletions on chromosome 10 are seen in both benign and higher-grade meningiomas and intracranial malignancies associated with radiotherapy. Immunohistochemical studies were performed to detect the protein product of the NF2 gene, merlin, in the four irradiated VS. NF2 staining was not observed. This study represents the first microsatellite and immunohistochemical analysis of recurrent VS following radiation therapy. Our preliminary observations suggest an alternative mechanism of NF2 inactivation that may correlate with radioresistance in VS.

A Mouse Model Recapitulating Molecular Features of Human Mesothelioma

Malignant mesothelioma has been linked to asbestos exposure and generally has a poor prognosis because it is often diagnosed in advanced stages and is refractory to conventional therapy. Human malignant mesotheliomas accumulate multiple somatic genetic alterations, including inactivation of the NF2 and CDKN2A/ARF tumor suppressor genes. To better understand the significance of NF2 inactivation in malignant mesothelioma and identify tumor suppressor gene alterations that cooperate with NF2 loss of function in malignant mesothelioma pathogenesis, we treated Nf2 (+/-) knockout mice with asbestos to induce malignant mesotheliomas. Asbestos-exposed Nf2 (+/-) mice exhibited markedly accelerated malignant mesothelioma tumor formation compared with asbestos-treated wild-type (WT) littermates. Loss of the WT Nf2 allele, leading to biallelic inactivation, was observed in all nine asbestos-induced malignant mesotheliomas from Nf2 (+/-) mice and in 50% of malignant mesotheliomas from asbestos-exposed WT mice. For a detailed comparison with the murine model, DNA analyses were also done on a series of human malignant mesothelioma samples. Remarkably, similar to human malignant mesotheliomas, tumors from Nf2 (+/-) mice showed frequent homologous deletions of the Cdkn2a/Arf locus and adjacent Cdkn2b tumor suppressor gene, as well as reciprocal inactivation of Tp53 in a subset of tumors that retained the Arf locus. As in the human disease counterpart, malignant mesotheliomas from the Nf2 (+/-) mice also showed frequent activation of Akt kinase, which plays a central role in tumorigenesis and therapeutic resistance. Thus, this murine model of environmental carcinogenesis faithfully recapitulates many of the molecular features of human malignant mesothelioma and has significant implications for the further characterization of malignant mesothelioma pathogenesis and preclinical testing of novel therapeutic modalities.

NF2: the wizardry of merlin.

Neurofibromatosis type II (NF2) is an autosomal dominant cancer syndrome characterized by the formation of tumors of the nervous system, particularly schwannomas and meningiomas. The NF2 gene is also implicated in the development of sporadic schwannomas and meningiomas, as well as tumor types seemingly unrelated to the NF2 disorder, such as malignant mesotheliomas. Inactivation of NF2 occurs by a "two-hit" mechanism, as proposed by Al Knudson, and the NF2 gene behaves as a classical tumor suppressor gene. The NF2 gene product, merlin, exhibits homology with the ezrin-radixin-moesin family of membrane-cytoskeleton-linking proteins. During the past several years, there has been intensive investigation aimed at elucidating the mechanisms underlying merlin's functions. In this review, we summarize the involvement of NF2 inactivation in tumorigenesis. We also discuss observations implicating merlin in cell motility and cell proliferation, with a focus on recent findings linking merlin to Rac signaling.

Frequent loss of 1p32 region but no mutation of the p18 tumor suppressor gene in meningiomas.

After chromosome 22 and NF2 inactivation, the loss of chromosome 1p is one of the most frequent abnormalities encountered in meningiomas. However the putative tumor suppressor gene located on 1p inactivated in meningiomas has still to be identified. We screened 68 meningiomas for LOH on chromosome 22 and 1. We found 34 LOH on the NF2 region on chromosome 22 (50%) and 19 LOH on 1p (28%), 16 being associated with loss of chromosome 22. Partial deletions delimited a candidate region located between D1S234 and D1S2797. The p18INK4C tumor suppressor gene, a member of the genes family coding for inhibitors of cyclin-dependent kinases, is located in this region. To determine whether p18 is involved in development of meningiomas, we performed a mutation analysis of the p18 gene and a search for homozygous deletion in the 19 meningiomas with 1p loss. Sequencing analysis of the p18 gene revealed one polymorphism, but no somatic mutations and no homozygous deletions were found. These results confirm that the loss of chromosome 1p32 is a frequent feature in meningiomas, however the p18 tumor suppressor gene which is located in this region, does not seem to be involved.

Frequent mutations of NF2 and allelic loss from chromosome band 22q12 in malignant mesothelioma: Evidence for a two‐hit mechanism of NF2 inactivation

We previously reported NF2 mutations in malignant mesothelioma (MM) cell lines and corresponding primary tumors. We have now generated polyclonal antibodies that specifically recognize the C-terminus of the NF2 protein. Western blot analysis was performed on 25 MM cell lines, 14 of which showed no NF2 expression. Single-strand conformation polymorphism and DNA sequence analyses revealed NF2 mutations in each of these 14 cell lines. To explore the mechanism of inactivation of NF2, loss of heterozygosity analysis was performed with two microsatellite markers located in the vicinity of the NF2 locus in chromosome band 22q12. Eighteen of the 25 cell lines (72%) showed losses at one or both loci tested. All cases exhibiting mutation and/or aberrant expression of NF2 showed allelic losses, suggesting that inactivation of NF2 in MM occurs via a two-hit mechanism. Genes Chromosomes Cancer 24:238–242, 1999. © 1999 Wiley-Liss, Inc.

Frequent mutations of NF2 and allelic loss from chromosome band 22q12 in malignant mesothelioma: Evidence for a two-hit mechanism ofNF2 inactivation

We previously reported NF2 mutations in malignant mesothelioma (MM) cell lines and corresponding primary tumors. We have now generated polyclonal antibodies that specifically recognize the C-terminus of the NF2 protein. Western blot analysis was performed on 25 MM cell lines, 14 of which showed no NF2 expression. Single-strand conformation polymorphism and DNA sequence analyses revealed NF2 mutations in each of these 14 cell lines. To explore the mechanism of inactivation of NF2, loss of heterozygosity analysis was performed with two microsatellite markers located in the vicinity of the NF2 locus in chromosome band 22q12. Eighteen of the 25 cell lines (72%) showed losses at one or both loci tested. All cases exhibiting mutation and/or aberrant expression of NF2 showed allelic losses, suggesting that inactivation of NF2 in MM occurs via a two-hit mechanism.