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Merlin-Deficient Human Tumors Show Loss of Contact Inhibition And Volume 13 Number 12 December 2011 pp. 1101–1112 1101 www.neoplasia.com Merlin-Deficient Human Tumors Lu Zhou, Emanuela Ercolano, Sylwia Ammoun, M. Caroline Schmid, Magdalena A. Barczyk Show Loss of Contact Inhibition and Clemens Oliver Hanemann β and Activation of Wnt/ -Catenin Clinical Neurobiology, Peninsula College of Medicine Signaling Linked to the PDGFR/Src and Dentistry, University of Plymouth, Plymouth, UK and Rac/PAK Pathways1,2 Abstract Neurofibromatosis type 2 (NF2) is an inherited predisposition cancer syndrome characterized by the development of multiple benign tumors in the nervous system including schwannomas, meningiomas, and ependymomas. Using a disease model comprising primary human schwannoma cells, we previously demonstrated that adherens junctions (AJs) are impaired in schwannoma cells because of a ubiquitous, upregulated Rac activity. However, the mech- anism by which loss of contact inhibition leads to proliferation remains obscure in merlin-deficient tumors. In this study, we show that proliferative Wnt/β-catenin signaling is elevated as active β-catenin (dephosphorylated at serine 37 and threoine 41) localizes to the nucleus and the Wnt targets genes c-myc and cyclin D1 are upregulated in confluent human schwannoma cells. We demonstrate that Rac effector p21-activated kinase 2 (PAK2) is essential for the activation of Wnt/β-catenin signaling because depletion of PAK2 suppressed active β-catenin, c-myc, and cyclin D1. Most importantly, the link between the loss of the AJ complex and the increased proliferation in human schwannoma cells is connected by Src and platelet-derived growth factor receptor–induced tyrosine 654 phosphor- ylation on β-catenin and associated with degradation of N-cadherin. We also demonstrate that active merlin main- tains β-catenin and N-cadherin complex at the plasma membrane through direct regulation. Finally, we demonstrate that phosphorylation of tyrosine 654 is critical for the increased proliferation in human schwannoma cells because overexpression of a Y654F mutant β-catenin reduces hyperproliferation of schwannoma cells. We suggest a model that these pathways are coordinated and relevant for proliferation in merlin-deficient tumors. Neoplasia (2011) 13, 1101–1112 Introduction Neurofibromatosis type 2 (NF2) is an inherited predisposition cancer Abbreviations: ABC, active β-catenin, dephosphorylated on Ser37 and Thr41; AJ, syndrome characterized by the development of multiple benign tumors adherens junction; ERM, ezrin-radixin-moesin; NF2, neurofibromatosis type 2; NHERF1/ EBP50, Na+-H+ exchanger regulatory factor 1/ERM binding protein; PAK, p21-activated in the nervous system including schwannomas, meningiomas, and kinase; PDGFR, platelet-derived growth factor receptor ependymomas. NF2 affects 1 in 30,000 people and leads to significant Address all correspondence to: Clemens Oliver Hanemann, MD, PhD, Clinical Neuro- medical problems, namely, substantial morbidity and reduced life biology, Peninsula College of Medicine and Dentistry, University of Plymouth, The span. The tumor suppressor protein merlin, which belongs to the ezrin- John Bull Building, Tamar Science Park, Research Way, Plymouth PL6 8BU, United Kingdom. E-mail: oliver. [email protected] radixin-moesin (ERM) family, is the protein product of the NF2 gene. 1The authors thank the Dr. Hadwen Trust for the funding support and the Dr. Hadwen Loss of merlin also causes sporadic tumors, namely, all schwannomas, Trust for Humane Research, the UK’s leading medical research charity funding exclu- a proportion of ependymomas, and most meningiomas [1]. Like other sively nonanimal research techniques to replace animal experiments. There are no con- – flicts of interest by any of the coauthors. ERM proteins, merlin can form a C-terminal tail FERM (Band 4.1, ezrin, 2This article refers to supplementary materials, which are designated by Figures W1 radixin, and moesin) interaction and switch between open and closed and W2 and are available online at www.neoplasia.com. conformation through changing phosphorylation status on serine 518. Received 28 July 2011; Revised 17 October 2011; Accepted 27 October 2011 Merlin localizes to the cortical cytoskeleton [2] and the nucleus [3]. At Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1522-8002/11/$25.00 the plasma membrane, merlin exerts inhibitory effects by regulating DOI 10.1593/neo.111060 1102 Dissecting Wnt/β-catenin Signaling in Human Schwannoma Zhou et al. Neoplasia Vol. 13, No. 12, 2011 growth factor receptors, integrins, and downstream signaling pathways, schwannoma cells [15], is the first step of β-catenin translocation from for example, Rac/PAK/JNK, integrins/Src/FAK, PI3K/Akt, mTOR, and the AJs to the cytoplasm [16]. In the absence of Wnt signaling, a de- Raf/MEK/ERK pathways [4–10]. In the nucleus, merlin suppresses construction complex comprising axin, GSK-3, and APC is formed, tumorigenesis by binding to the E3 ubiquitin ligase CRL4DCAF1, inhib- leading to the proteolytic degradation of β-catenin. Nevertheless, while iting its activity [3]. Wnt is present, serine 37 and threoine 41 on cytoplasmic β-catenin are Loss of contact inhibition is a hallmark of merlin-deficient tumors. dephosphorylated and β-catenin is shuttled into the nucleus [17]. Nu- The potential mechanism for this has been investigated by analyzing the clear translocation of β-catenin is dependent on the phosphorylation of relationship between merlin and Rac/PAK, CD44, Paxillin, epidermal Ser191 and Ser605 mediated by the active Rac1/JNK2 pathway; muta- growth factor receptor, and other growth factor receptors [5,8,11,12]. tions of these residues significantly affect β-catenin nuclear accumula- Although a colocalization and a physical interaction between merlin tion and Wnt signaling [18]. In the nucleus, β-catenin will then interact and the core members of the adherens junction (AJ) complex, namely, with the TCF/LEF family of transcription factors and activate Wnt cadherins and catenins, had been observed [13], it is still unclear how transcription target genes, for example, cyclin D1 and c-myc [14]. merlin directly inhibits proliferation by regulating cadherins and catenins. In a recent study, Wnt/β-catenin signaling, which was measured The Wnt signaling transduction pathway plays an important role by the expressions of Wnt target genes and TCF activity, was found in tumorigenesis [14]. The stability of β-catenin, the core player of to be significantly increased in NF2 knockout mouse embryonic fibro- the canonical Wnt pathway, is strictly regulated in the cell cytoplasm. blast in confluent cell cultures, which was reduced by knockdown or Tyrosine phosphorylation, which was detected on β-catenin in inhibition of Rac1 [19]. The increase in Wnt signaling had also Figure 1. Wnt signaling is elevated in human schwannoma cells. (A) Western blot analyses were carried out to compare the levels of β-catenin between Schwann and schwannoma cells. RhoGDI was used as a loading control between Schwann and schwannoma cells. No significant difference (ns) was observed after quantification and statistical analysis (n = 5). Error bars represent the mean ± SEM. (B) β-Catenin localizes in the nucleus of schwannoma but not Schwann cells. Confluent and 24-hour starved human Schwann (NF2+/+) and schwannoma cells (NF2−/−) were immunostained with DAPI (nuclear marker) and β-catenin. Scale bar, 20 μm. (C) ABC and Wnt target genes are elevated in schwannoma. Western blot analyses were carried out to compare the level of ABC, c-myc, and cyclin D1. (D) The nuclear portion of β-catenin in schwannoma is ABC. Immunostaining was carried out for ABC, DAPI, and cytoskeleton marker F-actin in Schwann and schwannoma cells. Scale bar, 10 μm. Neoplasia Vol. 13, No. 12, 2011 Dissecting Wnt/β-catenin Signaling in Human Schwannoma Zhou et al. 1103 Figure 2. Knockdown PAK2 suppresses Wnt/β-catenin signaling. Schwannoma cells were infected with shRNA encoding a sequence targeting human PAK2 (sh-PAK2a) or a matched scramble sequence (Control). Western blot analyses were carried out to compare the levels of pJNK, ABC, c-myc, cyclin D1, and β-catenin between sh-PAK2a and control. Densities of bands were quantified and compared. Error bars represent the mean ± SEM. n = 3. *P < .05. **P < .01. ns indicates no significant difference. pJNK, ABC, c-myc, and cyclin D1 were significantly reduced after knocking down PAK2. been correlated with merlin deficiency in human malignant gliomas nerve donors were used in this research. Isolation and culturing were [20]. In human primary schwannoma cells, increased activity of carried out as previously described [22]. Briefly, cells were cultured in Rac1/PAK/JNK [7,15,21] and colocalization of phosphorylated tyro- proliferation medium and grown on poly-L-lysine/laminin–coated six- sine (antibody) and β-catenin [15] prompted us to examine the fate well plates (Greiner, Bio One, Stonehouse, United Kingdom) or eight- and roles of β-catenin and related growth factor signaling in schwannoma well permanox chamber slides (Lab-Tek, Nunc, Wiesbaden, Germany). cells. More importantly, the mechanism by which merlin loss leads to HEK293T cells were cultured in standard conditions. All experiments increased Wnt/β-catenin in NF2 was investigated. We show that in- were performed on starved confluent cells unless otherwise stated, and all creased nuclear β-catenin signaling is initiated at the AJs and is regulated experiments were repeated with cells from at least three different patients. by tyrosine kinase receptors
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