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YAP signaling has emerged as an important signaling pathway involved in several normal and pathological processes. While main upstream effectors regulating its activity have been extensively studied, the interplay with other cellular processes has been far less analyzed. Here, we identified the LAMTOR complex as a new important regulator of YAP signaling. We uncovered that p18/LAMTOR1 is required for the recycling of Src on late endosomes to the cell periphery, and consequently to activate a signaling cascade that eventually controls YAP nuclear shuttling. Moreover, p18/LAMTOR1 positives late endosomes distribution is controlled by β1 integrins, extracellular matrix stiffness and cell contractility. This likely relies on the targeting of microtubules to β1 positive focal adhesion via ILK. Altogether our findings identify the late endosomal recycling pathway as a major regulator of YAP.

BACKGROUND: Clinical overlap between neurofibromatosis type 2 (NF2), schwannomatosis, and meningiomatosis can make clinical diagnosis difficult. Hence, molecular investigation of germline and tumor tissues may improve the diagnosis. METHODS: We present the targeted next-generation sequencing (NGS) of NF2, SMARCB1, LZTR1, SMARCE1, and SUFU tumor suppressor genes, using an amplicon-based approach. We analyzed blood DNA from a cohort of 196 patients, including patients with NF2 (N = 79), schwannomatosis (N = 40), meningiomatosis (N = 12), and no clearly established diagnosis (N = 65). Matched tumor DNA was analyzed when available. Forty-seven NF2-/SMARCB1-negative schwannomatosis patients and 27 NF2-negative meningiomatosis patients were also evaluated. RESULTS: A NF2 variant was found in 41/79 (52%) NF2 patients. SMARCB1 or LZTR1 variants were identified in 5/40 (12.5%) and 13/40 (∼32%) patients in the schwannomatosis cohort. Potentially pathogenic variants were found in 12/65 (18.5%) patients with no clearly established diagnosis. A LZTR1 variant was identified in 16/47 (34%) NF2/SMARCB1-negative schwannomatosis patients. A SMARCE1 variant was found in 3/39 (∼8%) meningiomatosis patients. No SUFU variant was found in the cohort. NGS was an effective and sensitive method to detect mutant alleles in blood or tumor DNA of mosaic NF2 patients. Interestingly, we identified a 4-hit mechanism resulting in the complete NF2 loss-of-function combined with SMARCB1 and LZTR1 haploinsufficiency in two-thirds of tumors from NF2 patients. CONCLUSIONS: Simultaneous investigation of NF2, SMARCB1, LZTR1, and SMARCE1 is a key element in the differential diagnosis of NF2, schwannomatosis, and meningiomatosis. The targeted NGS strategy is suitable for the identification of NF2 mosaicism in blood and for the investigation of tumors from these patients.

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.

The NF2 gene product, merlin/schwannomin, is a cytoskeleton organizer with unique growth-inhibiting activity in specific cell types. A narrow spectrum of tumors is associated with NF2 deficiency, mainly schwannomas and meningiomas, suggesting cell-specific mechanisms of growth control. We have investigated merlin function in mouse Schwann cells (SCs). We found that merlin regulates contact inhibition of proliferation by limiting the delivery of several growth factor receptors at the plasma membrane of primary SCs. Notably, upon cell-to-cell contact, merlin downregulates the membrane levels of ErbB2 and ErbB3, thus inhibiting the activity of the downstream mitogenic signaling pathways protein kinase B and mitogen-activated protein kinase. Consequently, loss of merlin activity is associated with elevated levels of ErbB receptors in primary SCs. We also observed accumulation of growth factor receptors such as ErbB2 and 3, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor in peripheral nerves of Nf2-mutant mice and in human NF2 schwannomas, suggesting that this mechanism could play an important role in tumorigenesis.

The Ras proteins play a central role in regulating cell growth and their mutation can lead to abnormal proliferation. To analyse the potential link betwen AP1 activity, encoded by members of the jun and fos gene families, and Ras-mediated cellular transformation, we have studied several NIH3T3 clones which overexpress the Ha-Ras or Ki-Ras oncogenes. These transformed fibroblasts accumulated higher levels of cJun, JunB, Fra1 and Fra2 proteins relative to their normal counterparts. They also displayed increased AP1 DNA binding activity which was predominantly composed of cJun and Fra1 containing dimers. Following serum stimulation of Ras clones, the elevated levels of cJun and Fral remained steady, while the induction of JunB and Fra2 was partially attenuated. Moreover, deregulated Ras signaling resulted in a complete loss of the serum inducibility of cFos and FosB. Ectopic co-expression of cJun and Fra1 in NIH3T3 fibroblasts led to a transformed phenotype, attenuation of cFos serum inducibility, increased AP1 activity and Cyclin D1 accumulation, all characteristics of oncogenic Ras expressing cells. These results demonstrate that cJun and Fra1 are crucial mediators of the Ras-transformation process.

We have analysed the different Jun and Fos proteins as NIH3T3 fibroblasts pass from exponential growth to quiescence and during the first 24 h after their re-entry into the cell cycle following serum stimulation. We show that these proteins can be divided into 3 subgroups based on their pattern of expression. The first contains c-Jun, Jun-D and Fra-2 which are expressed at high level in cycling cells and are only mildly induced by serum. The second contains Jun-B, c-Fos, Fos-B and deltaFos-B whose levels are low in cycling cells but increase strongly and rapidly after stimulation by serum. The third group contains only Fra-1, which is absent from cycling cells and behaves as a delayed early response protein after serum stimulation. AP-1 binding activity is low both in cycling and quiescent fibroblasts but increases after stimulation by serum with kinetics matching the induction of the various Jun and Fos proteins. Antibody supershift analyses demonstrate that the composition of AP-1 binding activity reflects the relative abundance of each Jun and Fos protein. Furthermore, the state of post-translational modification varies continuously for all of the AP-1 proteins as growth conditions change. These data indicate that AP-1 activity during the G0-G1 transition is finely regulated and complex, involving changes both in protein expression and in posttranslational modification.

The lack of neurofibromatosis 2 tumor suppressor protein merlin leads to the formation of nervous system tumors, specifically schwannomas and meningiomas. Merlin is considered to act as a tumor suppressor at the cell membrane, where it links transmembrane receptors to the actin cytoskeleton. Several tumor suppressors interact with another component of the cytoskeleton, the microtubules, in a regulated manner and control their dynamics. In this work, we identify merlin as a novel microtubule-organizing protein. We identify two tubulin-binding sites in merlin, one residing at the N-terminal FERM-domain and another at the C-terminal domain. Merlin's intramolecular association and phosphorylation of serine 518 regulate the interaction between merlin and tubulin. Analysis of cultured glioma cells indicates colocalization between merlin and microtubules especially during cell division. In primary mouse Schwann cells only minor colocalization at the cell periphery of interphase cells is seen. However, these cells drastically change their microtubule organization upon loss of merlin indicating a functional association of the proteins. Both in vitro assays and in vivo studies in Schwann cells indicate that merlin promotes tubulin polymerization. The results show that merlin plays a key role in the regulation of the Schwann cell microtubule cytoskeleton and suggest a mechanism by which loss of merlin leads to cytoskeletal defects observed in human schwannomas.

Barsh, G.S. (2006). Regulation of pigment type switching by agouti, melanocortin signaling, attractin and mahoganoid. In The Pigmentary System, 2nd edn. J.J. Nordlund, R.E. Boissy, V.J. Hearing, R.A. King, W.S. Oetting, and J.P. Ortonne, eds (Massachusetts: Blackwell Publishing), pp. 395–409. Candille, S.I., Kaelin, C.B., Cattanach, B.M., Yu, B., Thompson, D.A., Nix, M.A., Kerns, J.A., Schmutz, S.M., Millhauser, G.L., and Barsh, G.S. (2007). A b-defensin mutation causes black coat color in domestic dogs. Science 318, 1418–1423. Chakrabarti, O., and Hegde, R.S. (2009). Functional depletion of mahogunin by cytosolically exposed prion protein contributes to neurodegeneration. Cell 137, 1136–1147. Hida, T., Wakamatsu, K., Sviderskaya, E.V. et al. (2009). Agouti protein, mahogunin, and attractin in pheomelanogenesis and melanoblast-like alteration of melanocytes: a cAMP-independent pathway. Pigment Cell Melanoma Res. 22, 623–634. Jiao, J., Kim, H.Y., Liu, R.R., Hogan, C.A., Sun, K., Tam, L.M., and Gunn, T.M. (2009). Transgenic analysis of the physiological functions of Mahogunin Ring Finger-1 isoforms. Genesis 47, 524–534.

Sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis in its absence. We have investigated the pattern of expression of the Jun and Fos family of transcription factors in dying sympathetic neurons using antibodies specific for each family member. When sympathetic neurons are deprived of NGF, the level of c-Jun protein significantly increases, whereas the levels of the other members of the Jun and Fos family remain relatively constant. c-Jun also becomes more phosphorylated, probably on its amino terminal transactivation domain. When microinjected into sympathetiic neurons, an expression vector for a c-Jun dominant negative mutant protects them against NGF withdrawal-induced death, indicating that AP-1 activity is essential for neuronal cell death. Furthermore, overexpression of the full-length c-Jun protein is, in itself, sufficient to induce apoptosis in sympathetic neurons.

We have measured the level of junB mRNA in the B hybridoma cell line 7TD1, under interleukin-6 (IL-6) stimulation. IL-6 increases junB mRNA in a biphasic fashion. The first early-induced peak was transient and likely corresponds to the well documented typical junB mRNA, stimulated in response to numerous growth factors, including IL-6. At variance, the second peak which has never been reported previously, lasted several hours. As a consequence of its effect on junB mRNA, IL-6 stimulated, in a biphasic fashion, the nuclear accumulation of the JunB protein. In this study, we demonstrated that IL-6 regulation occurred exclusively at the transcriptional level and that the bimodal increase of junB mRNA and JunB protein can be accounted for by a biphasic stimulation of junB transcription. Furthermore, our data point to two major differences between the mechanism of control of the early and the late IL-6-induced junB transcription waves. First, cycloheximide strongly potentiated the transcription of the second wave, whereas it failed to affect the early-induced burst. Second, tyrphostin, a tyrosine kinase inhibitor, impaired the expression of the first but not the second junB mRNA peak. Conversely, genistein, another tyrosine kinase inhibitor, totally abolished the expression of the second peak of junB mRNA whereas it did not affect the expression of the first peak. Altogether these data indicate that, in 7TD1 cells, IL-6 controls junB transcription in a biphasic fashion by means of two separate transduction pathways.

As NIH 3T3 fibroblasts become quiescent, the level of c-Jun protein decreases while JunD accumulates. When resting cells are stimulated with fresh serum, nuclear-localized JunD is rapidly degraded, followed by resynthesis of both c-Jun and JunD later in G1. Overexpression of JunD results in slower growth and an increase in the percentage of cells in G0/G1 while c-Jun overexpression produces larger S/G2 and M phase populations. In addition, JunD partially suppresses transformation by an activated ras gene whereas c-Jun cooperates with ras to transform cells. These data indicate that two closely related transcription factors can function in an opposing manner.

Lovastatin-induced inhibition of renal epithelial tubular cell proliferation involves a p21 ras activated, AP-1-dependent pathway. Proliferation of tubular epithelial cells underlies the development of cystic lesions and the subsequent impairment of renal function after renal mass reduction. The effect of HMG CoA reductase inhibitors (HRI) on cell proliferation was investigated in rat renal proximal tubular epithelial cells in primary culture. Treatment of renal tubular epithelial cells with three different HRI reduced fetal calf serum (FCS)-induced [ 3 H]-thymidine incorporation (IC 50 values were 0.7 µ M, 1.7 µ M, and 1.6 µ M for simvastatin, lovastatin, and compactin, respectively), and lovastatin blocked BrdUrd incorporation, as assessed by immunocytochemical studies. The proliferative effect of epidermal growth factor (EGF) was similarly abolished by lovastatin. The effect of lovastatin (1 µ M) was prevented by 100 µ M mevalonate, 5 µ M farnesyl-pyrophosphate and 5 µ M geranylgeranyl-pyrophosphate (in percent of control value, 31% vs. 102%, 60%, and 82%, respectively) while cholesterol and other products of the mevalonate pathway were inactive. Immunoblot analysis showed that lovastatin decreased membrane-bound p21 ras and inhibited FCS-induced c-fos and c-jun protein expression. Furthermore, electrophoretic mobility shift assay demonstrated the functional impairement of AP-1 DNA binding activity in lovastatin-treated cells. In conclusion, these results demonstrate that HRI are antiproliferative in epithelial tubule cells and that this effect is exerted, at least in part, via inhibition of the p21 ras -activated and AP-1 dependent mitogenic cascade.