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

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 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 PDGFRs and signaling pathways such as MAPK, Rac/PAK, JNK, or β-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 that promote their growth. Using a set of more than 40 human schwannomas, we have analyzed the status of several major signaling pathways that were previously shown to be regulated by merlin. First, using receptor tyrosine kinase (RTK) arrays and Western blotting, we have identified which RTKs are the most frequently activated in schwannomas and could therefore represent a potential therapeutic target. Then, RPPA was used on our tumor set to measure the expression and activity of 35 proteins comprising the major mitogenic signaling pathways. Our results showed that expression levels of YAP and several of its target genes, notably several receptors identified through RTK arrays, are linked to proliferation. However, immunohistochemical analysis revealed a strong variability of protein expression from tumor to tumor, suggesting that the response to targeted chemotherapy would likely be highly variable. Altogether, our work identifies a signaling network associated with YAP protein and linked to schwannoma cell proliferation that could represent a set of valuable therapeutic targets. Statement of translational relevance Patients affected by the familial syndrome Neurofibromatosis type 2 develop multiple tumors when the NF2 suppressor gene is inactivated. Schwannomas are the most frequent ones and the number of chemotherapeutic options is yet very limited. Hence, the identification of potential therapeutic targets is a major objective of the research effort on this tumor type. In this study, we have combined several proteomic approaches to investigate the expression and activity of mitogenic signaling pathways regulated by NF2. Our work shows that proliferation of tumor cells is correlated to the activation of a signaling network linked to the Hippo effector Yap and several of its target genes. Hence, our study provides evidences that Yap as well as its target genes PDGFRs, Her3, Her2 and Axl represent potentially new therapeutic targets for the treatment of schwannomas.