Proteomic screening identifies a YAP-driven signaling network linked to tumor cell proliferation in human schwannomas

Neuro-Oncology Neuro-Oncology 16(9), 1196– 1209, 2014 doi:10.1093/neuonc/nou020 Advance Access date 20 February 2014 Proteomic screening identifies a YAP-driven signaling network linked to tumor cell proliferation in human schwannomas Alizée Boin†, Anne Couvelard†, Christophe Couderc, Isabel Brito, Dan Filipescu, Michel Kalamarides, Pierre Bedossa, Leanne De Koning, Carine Danelsky, Thierry Dubois, Philippe Hupé, Daniel Louvard, and Dominique Lallemand Corresponding Author: Dominique Lallemand, PhD, UMR144, Institut Curie, 26 Rue d’Ulm, 75248 Paris Cedex 05, France (). † These authors contributed equally to this work. Background. Inactivation of the NF2 gene predisposes to neurofibromatosis type II and the development of schwannomas. In vitro studies have shown that loss of NF2 leads to the induction of mitogenic signaling mediated by receptor tyrosine kinases (RTKs), MAP kinase, AKT, or Hippo pathways. The goal of our study was to evaluate the expression and activity of these signaling pathways in human schwannomas in order to identify new potential therapeutic targets. Methods. Large sets of human schwannomas, totaling 68 tumors, were analyzed using complementary proteomic approaches. RTK arrays identified the most frequently activated RTKs. The correlation between the expression and activity of signaling pathways and proliferation of tumor cells using Ki67 marker was investigated by reverse-phase protein array (RRPA). Finally, immunohistochemistry was used to evaluate the expression pattern of signaling effectors in the tumors. Results. We showed that Her2, Her3, PDGFRß, Axl, and Tie2 are frequently activated in the tumors. Furthermore, RRPA demonstrated that Ki67 levels are linked to YAP, p-Her3, and PDGFRß expression levels. In addition, Her2, Her3, and PDGFRß are transcriptional targets of Yes-associated protein (YAP) in schwannoma cells in culture. Finally, we observed that the expression of these signaling effectors is very variable between tumors. Conclusions. Tumor cell proliferation in human schwannomas is linked to a signaling network controlled by the Hippo effector YAP. Her2, Her3, PDGFRß, Axl, and Tie2, as well as YAP, represent potentially valuable therapeutic targets. However, the variability of their expression between tumors may result in strong differences in the response to targeted therapy. Keywords: Neurofibromatosis type 2, proteomic, schwannoma, signaling, YAP. Biallelic inactivation of the NF2 tumor suppressor gene leads to the development of intracranial tumors such as schwannomas, meningiomas, and ependymomas. These lesions can be sporadic or develop in the context of an inherited familial disease called neurofibromatosis type 2. Patients who are born heterozygous for NF2 are predisposed to the development of multiple tumors upon subsequent loss of the second allele.1 To this day, surgery and radiotherapy remain the most frequent options for treatment. The use of chemotherapy has long been hindered by the lack of clearly identified therapeutic targets. Since the discovery of the NF2 gene in 1993,2 a major effort has been made to understand how merlin, the NF2 gene product, regulates cell proliferation and tumor growth. Several proteins playing key roles in these processes have emerged as candidate therapeutic targets. The inactivation of NF2 was shown to trigger the activation of Ras and Rac and the stimulation of downstream mitogenic p42/44 MAPK and PAK1/2 pathways.3,4 It has also been demonstrated that loss of merlin expression induces the activity and surface expression of various receptor tyrosine kinases (RTKs). In the latter case, it is due to an increase of their transport to the plasma Received 29 January 2014; accepted 29 January 2014 # The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: . 1196 Centre National de la Recherche Scientifique, Institut Curie, Paris, France (A.B., C.C., D.Lo., D.La.); Institut National de la Santé et de la Recherche Médicale, Paris, France (I.B., P.H.); Mines ParisTech, Fontainebleau, France (P.H.); Breast Cancer Biology Group, Institut Curie, Paris, France (T.D.); Reverse Phase Protein Array Platform, Institut Curie, Paris, France (C.D., L.D.K.); Centre National de la Recherche Scientifique, Institut Curie, Paris, France (D.F.); Department of Neurosurgery, Assistance Publique-Hôpitaux de Paris, Hopital Beaujon, Clichy, France (M.K.); Unité Institut National de la Santé et de la Recherche Médicale, Fondation Jean Dausset, Paris, France (M.K.); Pathology Department Beaujon-Bichat, AP-HP, Hôpital Bichat, Paris, France (A.C.); Université Paris Diderot, Sorbonne Paris Cité, Paris, France (A.C.); Pathology Department Beaujon-Bichat, AP-HP, Hôpital Beaujon, Clichy, France (P.B.); Université Paris Diderot, Sorbonne Paris Cité, Paris, France (M.K.) Boin et al.: Proteomic study of signaling events in human schwannomas membrane,5 – 7 leading to stimulation of promitogenic signaling pathways. Recently, a new function for merlin in the nucleus was discovered.8 In this compartment, the binding of merlin to D-Caf1 abolishes its ubiquitin ligase activity and leads to the inhibition of proliferation. Finally, the regulation of the Hippo signaling pathway by merlin has emerged in recent years as a major mechanism controlling cell proliferation and survival in various organisms and tissues.9,10 Indeed, in several models, loss of merlin expression leads to nuclear accumulation of Yes-associated protein (YAP), which is the major effector of the Hippo pathway.11,12 There, it stimulates transcription of a wide set of promitogenic and anti-apoptotic target genes.13 Hence, the growth advantage conferred by the loss of NF2 appears to be the consequence of an accumulation of distinct signaling dysfunctions. The relevance of these mechanisms of growth control by merlin has also been tested on tumor cell cultures derived from human biopsies. Receptors such as IGF1R, Axl, or PDGFRß and signaling pathways such as MAPK, Rac/PAK, JNK, or b-catenin were shown to be activated in response to merlin loss of expression.14 – 16 However, the tumor cell cultures always represent a simplified model of their tumor of origin. In this context, evaluation of the expression and activity of signaling pathways in surgical biopsies represents a necessary complement to the previous approaches. Immunohistological evaluation of the expression and activity of signaling proteins in tumor sections has provided important information on the biology of NF2-related tumors.17 – 19 Nevertheless, new proteomic approaches such as reverse phase protein arrays (RPPA) allow more precise means for quantifying signaling protein expression and activity on large sets of biological samples.20 Therefore, combining different types of proteomic analysis for the study of tumors constitutes a powerful approach for identifying the signaling events th (...truncated)