YAP Mediates Tumorigenesis in Neurofibromatosis Type 2 by Promoting Cell Survival and Proliferation Through a COX-2−EGFR Signaling Axis

Published OnlineFirst May 23, 2016; DOI: 10.1158/0008-5472.CAN-15-1144 Cancer Molecular and Cellular Pathobiology Research

YAP Mediates Tumorigenesis in Neuroﬁbromatosis Type 2 by Promoting Cell Survival and Proliferation through a COX-2–EGFR Signaling Axis William Guerrant1, Smitha Kota1, Scott Troutman1, Vinay Mandati1, Mohammad Fallahi2, Anat Stemmer-Rachamimov3, and Joseph L. Kissil1

Abstract

The Hippo–YAP pathway has emerged as a major driver of survival, proliferation, and tumor growth in vivo.Moreover, tumorigenesis in many human cancers. YAP is a transcrip- YAP promotes transcription of several targets including tional coactivator and while details of YAP regulation are PTGS2, which codes for COX-2, a key enzyme in prostaglan- quickly emerging, it remains unknown what downstream din biosynthesis, and AREG, which codes for the EGFR ligand, targets are critical for the oncogenic functions of YAP. To amphiregulin. Both AREG and prostaglandin E2 converge to determine the mechanisms involved and to identify disease- activate signaling through EGFR. Importantly, treatment with relevant targets, we examined the role of YAP in neuroﬁbro- the COX-2 inhibitor celecoxib signiﬁcantly inhibited the matosis type 2 (NF2) using cell and animal models. We found growth of NF2-null Schwann cells and tumor growth in a that YAP function is required for NF2-null Schwann cell mouse model of NF2. Cancer Res; 76(12); 1–13. 2016 AACR.

Introduction refs. 5, 6). Additional evidence for an oncogenic role of YAP in human tumors stems from findings demonstrating ampli- The Hippo–YAP signaling pathway has emerged as a major fication of genomic region 11q22, to which YAP localizes, in driver of tumorigenesis and metastasis in a wide spectrum of breast cancer and significant upregulation of YAP expression human cancers (1–3). The core of the pathway is composed of a in breast, ovarian, lung, pancreatic, colorectal, and liver can- well-defined kinase cascade composed of the MST1/2 kinases that cers (7, 8). More recent studies have shown that YAP can form a complex with the scaffold protein WW45 and phosphor- function as an oncogene in tumors that are addicted to KRAS. ylate the LATS1/2 kinases. Phosphorylated LATS1/2, in complex Specifically, in models of KRAS-addicted tumors (pancreatic with Mob1, bind and phosphorylate YAP, a transcriptional co- and lung adenocarcinoma), the inhibition of KRAS leads to activator. The phosphorylation of YAP creates a binding site for cell death, which can be rescued by YAP activation (9, 10). 14-3-3 and this prevents p-YAP from entering into the nucleus Finally, genetic evidence for an oncogenic role for YAP in where it can form transcriptionally active complexes with TEADs human cancer comes from two diseases, uveal melanoma and and other transcription factors to drive the expression of propro- neurofibromatosis type 2 (NF2). In uveal melanoma, 80% of liferative or antiapoptotic genes such as CTGF, Cyr61, Axl, Myc, patients harbor mutations in the GNAQ (Gq) and GNA11 and BIRC5 (4). (G11) genes, which code for alpha subunits of heterotrimeric As a regulator of cell fate, proliferation, and death, YAP can G proteins. Previous work had indicated YAP can be activated function as an oncogene. Several examples exist showing that by mutated Gq/11 (11), and subsequently, it was found that YAP overexpression drives tumorigenesis, including mouse mutated Gq/11 oncogenic function is mediated via YAP, thus models in which liver-specific expression of an activated allele implicating YAP as a potential therapeutic target in uveal of YAP or knockout of the MST1 and MST2 alleles in the liver melanoma (12, 13). lead to development of hepatocellular carcinoma (HCC; NF2 is an inherited disorder with an incidence of approximately 1 in 30,000 births, caused by germline mutations of the NF2 1Department of Cancer Biology, The Scripps Research Institute, Jupi- gene. The disease is characterized mainly by development ter, Florida. 2Informatics Core, The Scripps Research Institute, Jupiter, of schwannomas of the eighth cranial nerve (14). The NF2 3 Florida. Department of Pathology, Massachusetts General Hospital, tumor suppressor gene encodes a 69-kDa protein called Merlin Boston, Massachusetts. thathasbeenshowntofunctionasaregulatorofmultiple Note: Supplementary data for this article are available at Cancer Research signaling pathways at the cell membrane and to possess nuclear Online (http://cancerres.aacrjournals.org/). functions. Merlin was originally shown to function upstream of Corresponding Author: Joseph L. Kissil, Scripps Research Institute, 130 Scripps Hippo in flies and subsequently in mammalian cells. A number Way, Jupiter, FL 33458. Phone: 561-228-2170; Fax: 561-228-2175; E-mail: of studies demonstrated that Merlin and YAP function antag- [email protected] onistically, including in vivo studies in which liver-specific doi: 10.1158/0008-5472.CAN-15-1144 knockout of Yap was sufficient to rescue HCC driven by inac- 2016 American Association for Cancer Research. tivation of the Nf2 gene (15).

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Mechanistic details of the function of Merlin have emerged eration Assay (Millipore) was used according to the manufac- from studies that demonstrated Merlin acts synergistically with a turer's instructions. Statistical significance was determined by a newly identified Hippo pathway component, Kibra, to promote two-tailed Student t test. Each condition at each time point LATS1/2 phosphorylation (16) and regulate the spatial organi- represents the mean of three experiments in triplicate for a total zation of Hippo pathway components at the cell membrane of 9 wells. bydirectlybindingtoLATS1/2andrecruitingittotheplasma membrane, where it is phosphorylated and activated by a MST- Determination of caspase activity WW45 complex (17). Merlin has also been shown to have a Measurement of caspase-dependent cell death was achieved nuclear function as an inhibitor of the E3 ubiquitin ligase through the use of the Caspase-Glo 3/7 assay following the CRL4DCAF1 (18). Recent studies suggest that CRL4DCAF1 pro- manufacturer's instructions (Promega). Briefly, cells were seed- motes YAP- and TEAD-dependent transcription by inhibition of ed into white, opaque 96-well culture plates at 1,500 cells per LATS1/2 in the nucleus, and analysis of patient samples indi- well and transfected with control or YAP siRNAs. Caspase-Glo cates that this pathway operates in NF2-mutant tumors (19). reagent was added at 24 or 48 hours and incubated at room Thus, while the evidence cited above strongly suggests that YAP temperature for 30 minutes, after which the luminescence was function is required downstream of NF2 loss of function in measured. tumorigenesis, the mechanisms underlying the requirement for YAP and for which downstream targets are critical to the onco- RNA-Seq genic functions of YAP remain unknown. To identify these SC4 cells were transfected with control or YAP SMARTpool mechanisms and identify disease-relevant targets, we employed siRNA for 48 hours, and total RNA was extracted using TRIzol a combination of cell-based and in vivo approaches. Our findings reagent. For analysis, the sequencing reads in color space were indicate that YAP function is required in NF2-null Schwann cells mapped to the mm9 genome using Tophat (23). The number of fi to promote cell survival through regulation of an EGFR signaling reads falling into each gene de ned in the RefSeq gene annota- fi axis via transcriptional regulation of prostaglandin endoperoxide tions was quanti ed using HTSeq-count (24). The DESeq soft- ware (24) was used to detect differentially expressed genes synthase 2 (COX-2) and prostaglandin E2 (PGE2) production. Importantly, our findings suggest that treatment with COX-2 between samples. Samples from three independent experiments fi inhibitors could prove beneficial in slowing the growth rates of were sequenced, combined, and analyzed to produce the nal NF2-associated schwannomas. DESeq data. The RNA-Seq data are publicly available through the NCBI GEO database with accession number GSE61528.

Materials and Methods RT-PCR Animal experiments RNAs were extracted using the Qiagen RNeasy Kit and reverse- All animal experiments complied with NIH guidelines and transcribed into cDNA with the SuperScript III Kit (Life Technol- were approved by The Scripps Research Institutional Animal ogies). qPCR was performed with SYBR Green (Applied Biosys- Care and Use Committee. A total of 5 104 SC4-Luc pLKO or tems). Relative gene expression between control and YAP-KD was SC4-Luc pLKO-YAP cells were injected intraneurally into calculated with the 2 DDCT method (25). For complete primer the sciatic nerves of NOD/SCID mice (6–8 weeks old). Tumor sequences, please see Supplementary Table S2. progression was monitored by bioluminescence imaging on an IVIS-200 system (Xenogen). Treatment was commenced after AREG ELISA detection of signal correlating to tumor sizes of 1 to 2 mm3 Cell media from SC4 and HEI-193 cells treated with the (total flux 106 photons/s). For drug treatment, celecoxib indicated siRNA's was collected and diluted 2-fold with provided (Cayman Chemical) was diluted in vehicle (22.2:66.6:11.2, assay buffer (Mouse AREG ELISA and Human AREG ELISA, ethanol:PEG300:water) to a final dose of 100 mg/kg and Raybiotech). Secreted AREG concentrations were assayed for three administered by oral gavage, daily. Control mice received independent experiments in triplicate, according to manufac- vehicle/DMSO mixture. turer's instructions. AREG concentrations were determined by comparing recorded absorbance readings to a standard curve of Cell lines diluted AREG. SC4 Nf2-null mouse Schwann cells and HEI-193 human NF2- mutant Schwann cells were obtained in 2010 and previously Results described (20, 21). HSC2l cells were obtained from the labora- YAP is required for NF2-null Schwann cell proliferation, tory of Dr. Margret Wallace (University of Florida) in 2015. These survival, and tumorigenesis cells are derived from normal human Schwann cells that were To examine the role of YAP in NF2-null Schwann cells, three cell TERT and CDK4R24C immortalized by expression of (Wallace, lines were used: SC4—Nf2-null mouse Schwann cells, HEI-193— manuscript in preparation). SC4-Luc cells were previously NF2-null schwannoma cells isolated from an NF2 patient, and described (22). SC4, HEI-193, and HSC2l cells were authenti- HSC2l cells—immortalized normal human Schwann cells in fi cated by short tandem repeat (STR) DNA pro ling (DDC Medical; which two shRNAs were used to knockdown the expression of March 2015). Merlin (Supplementary Fig. S1A). As expected, the loss of Merlin expression resulted in increased cell numbers over time (Supple- Cell proliferation and viability mentary Fig. S1B). Cell viability was determined by luminescent ATP-dependent Using two independent siRNA sequences and siRNA SMART- assay (CellTiter-Glo, Promega), according to manufacturer's pool, the expression of YAP was knocked down and effects on cell instructions. For measurement of proliferation, the BrdU Prolif- numbers were examined. A significant decrease in cell numbers

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was observed, with numbers reduced 1.5-fold in SC4 cells, 2-fold YAP regulates diverse transcriptional programs in Nf2-null for HEI-193 cells, and 2- to 2.5-fold for HSC2l cells, over the Schwann cells course of 72 hours, respectively (Fig. 1A and B, Supplementary Fig. To understand the specific contribution of YAP to the sur- S1C–S1E). Next, verteporfin, a known inhibitor of the YAP–TEAD vival of Nf2-null Schwann cells, an RNA sequencing approach interaction was used to pharmacologically test the requirement of wasusedtoidentifyspecific YAP-regulated genes. SC4 cells YAP in NF2-null Schwann cells (26). Treatment of SC4 and HEI- were treated with either nontargeting or pooled YAP siRNAs for 193 cells over 72 hours with 1 mmol/L verteporfin significantly 48 hours, followed by RNA isolation and sequencing. A num- reduced cell numbers by 3- to 8-fold over 72 hours, respectively ber of genes were identified as significantly regulated by YAP (Supplementary Fig. S1F and S1G). and classified on the basis of functional annotation including To determine whether the effects of YAP knockdown are due genes involved in regulation of matrix metalloproteases, pros- to reduced cell proliferation or increased cell death rates, we tanoid biosynthesis, fatty acid oxidation, and G-coupled recep- assessed BrdU incorporation in YAP-knockdown SC4 and HEI- tor signaling (Fig. 3A–C). We focused on PTGS2, which codes 193 cells. BrdU incorporation was slightly reduced over 72 for the COX-2 protein, an enzyme that functions as a catalytic hours, indicating that YAP knockdown has a relatively small, intermediary in the synthesis of prostaglandins from arachi- but reproducible, impact on cell proliferation (Fig. 1C and D). donic acid. PTGS2 was validated as a YAP target by RT-PCR in To assess the effects of YAP inhibition on cell death, we assessed SC4 and HEI-193 cells, as knockdown of YAP resulted in a 2- to the viability of SC4 and HEI-193 cells treated with verteporfin 3-fold reduction in PTGS2 transcription, confirming the RNA- in a dose-dependent manner. VP concentrations as low as 1 Seqresults(Fig.3D).Thetop12genesregulatedbyYAPalso mmol/L caused a 50% reduction in viability of both SC4 and included AREG, which has been previously reported as a YAP HEI-193 cells, as measured by an ATP-dependent luminescent target and codes for amphiregulin (AREG), an EGFR ligand assay (Fig. 1E and F). That inhibition of YAP leads to increased (Fig. 3B) (27). YAP knockdown in both SC4 and HEI-193 cells cell death rates is further corroborated by increased levels of resulted in 2- to 5-fold reduction in AREG transcription (Fig. apoptotic markers, cleaved caspase-3, caspase-7, and PARP in 3E). Examination of intracellular levels of COX-2 protein by SC4 cells infected with a vector expressing YAP shRNA (SC4 WesternblottinginthepresenceorabsenceofYAPindicates pLKO-YAP) compared with SC4 cells infected with a control that in response to YAP knockdown, COX-2 levels are drasti- shRNA vector (SC4 pLKO; Fig. 1G). Similarly, increased cas- cally reduced. (Fig. 4A). To assess levels of AREG, we analyzed pase-3/7 activity was detected in SC4 and HEI-193 cells in the cell culture media for secreted protein employing an ELISA which YAP expression was knocked down (Supplementary approach. A highly reproducible decrease in secreted AREG was Fig. S1H and S1I). observed in the culture media of SC4 and HEI-193 cells in To investigate the requirement of YAP for Nf2-null Schwann which YAP or AREG are knocked down (Fig. 4B and Supple- cell tumorigenesis, we first assessed the ability of cells with YAP mentary Fig. S3A). knockdown to form colonies in soft agar. SC4 pLKO and SC4 pLKO-YAP cells were plated at equal numbers, and colony COX-2 is a target of YAP required for growth of NF2-null formation was assessed after 14 days. The knockdown of YAP Schwann cells significantly impaired the ability of SC4 pLKO-YAP cells to COX-2 functions as a mediator of prostanoid synthesis, form colonies and reduced colony numbers by 2-fold (Fig. 2A including prostaglandin E2 (PGE2), previously shown to pro- and Supplementary Fig. S2A). Moreover, assessing colony size mote proliferation and survival of various types of tumor cells revealed a typical size of approximately 50 mmforSC4pLKO- (28). Thus, the identification and confirmation of AREG and YAP cells compared with approximately 200 mmfortheSC4 PTGS2 as targets of YAP-dependent transcription links two pLKO cells (Supplementary Fig. S2B). To assess the require- paracrine signaling mediators of cell survival to the Hippo– ment of YAP for Nf2-null Schwann cell tumorigenesis in vivo, YAP pathway in NF2-null Schwann cells. To assess the specific luciferase-expressing SC4 cells (SC4-Luc) were infected with role of COX-2 downstream of YAP in NF2-null schwannoma either a control vector (SC4-Luc pLKO) or a vector expressing cells, SC4 and HEI-193 cells were treated with control or COX-2 YAP shRNA (SC4-Luc pLKO-YAP) to generate SC4-Luc cells siRNA, and the effect on cell numbers was assessed (Fig. 4A, C, with stable YAP knockdown (Fig. 2B), which lead to inhibition and D and Supplementary Fig. S3B and S3C). In both cell lines, of SC4-Luc cell growth in culture (Fig. 2C). SC4-Luc pLKO and COX-2 knockdown reduced cell numbers by approximately SC4-Luc pLKO-YAP cells were orthotopically implanted into 50% at 72 hours, although this was not as strong as YAP the sciatic nerves of NOD/SCID mice, and schwannoma tumor knockdown (compare to Fig. 1A and B), implicating additional formation and growth were monitored via bioluminescent downstream effectors in mediating YAP activity. To assess the imaging. Mice were imaged every other day, starting on day 6, requirement for COX-2 activity in driving YAP-dependent and were sacrificed on day 20. Both control and YAP knock- increase in cell numbers, we also employed the COX-2 inhib- down cohorts developed measurable tumor signals within 8 to itor celecoxib, which significantly inhibited the growth of SC4 10 days postimplantation. However, compared with SC4-Luc cells in a dose-dependent manner (Fig. 4E). pLKO controls, YAP knockdown significantly inhibited the To determine whether the effects of YAP–COX-2 are mediated growth of the Nf2-null schwannomas in vivo (Fig. 2D–Fand through PGE2, SC4 pLKO-YAP cells were treated with exogenous Supplementary Fig. S2C). Subsequent ex vivo analysis of excised PGE2 in an attempt to rescue the phenotype conferred by YAP tumors indicated that tumors arising from SC4-Luc pLKO-YAP knockdown. PGE2 treatment was able to partially rescue the cells were significantly smaller than tumors in the control effects of YAP knockdown by approximately 30% at 72 hours cohort(Fig.2G).Takentogether,ourdataindicatethatYAP (Fig. 4F). These results indicate COX-2 and PGE2 are essential activity is required for the proliferation and survival of Nf2-null effectors of YAP activity that are required, at least in part, for Nf2- Schwann cells in vitro and tumor growth in vivo. null Schwann cell growth.

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A 14 Control siRNA 24 h 48 h 72 h 12 YAP Smartpool

– + ––++ 5 YAP Smartpool 10 Control siRNA +–– +–+ 8

YAP 6

Cells/well × 10 Cells/well 4

Vinculin 2 0 SC4 0 24 48 72 Time (h)

B 14 24 h 48 h 72 h Control siRNA 12 YAP siRNA #1

YAP siRNA #1 – + ––++ 5 10 Control siRNA +–– +–+ 8 YAP 6

Cells/well × 10 Cells/well 4 Vinculin 2 HEI-193 0 0 24 48 72 Time (h) C D 180 Control siRNA 180 Control siRNA YAP Smartpool YAP siRNA #1 160 160 YAP siRNA #2 140 140 P = N.S. P < 0.05 P < 0.001 P = N.S. P = N.S. P < 0.001 120 P < 0.01 P < 0.001 P < 0.001 120 100 100 80 80 60 60 % BrdU Incorporation % BrdU Incorporation 40 40 20 20 0 0 24 48 72 24 48 72 Time (h) Time (h) SC4 HEI-193

EF G 120 120 P < 0.05 P < 0.05 100 100

80 80

60 60 SC4 pLKOSC4 pLKO-YAP cl-Caspase-3

% Viability 40 % Viability 40 cl-Caspase-7 20 20 cl-PARP 0 0 Vinculin 0 0.5 1 2.5 5 0 0.5 1 2.5 5 Verteporfin (mmol/L) Verteporfin (mmol/L) SC4 HEI-193

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Given the partial rescue of the effects of YAP knockdown by Nf2-null Schwann cell proliferation and survival through an PGE2, we next assessed whether the combination of PGE2 and EGFR/AKT signaling pathway. AREG would result in a greater rescue of the YAP knockdown phenotype. While treatment with PGE2 or AREG alone led Promotion of NF2-null Schwann cell growth by YAP is mediated to only a partial rescue of the YAP knockdown phenotype, through COX-2 and AREG the combined actions of both targets rescued the phenotype, To determine the extent to which the effects of YAP on NF2- increasing cell numbers to levels comparable to control SC4- null Schwann cells are mediated by COX-2 and AREG, we pLKO cells (Fig. 4F–H).Importantly,treatmentofcontrolSC4- employed a gain-of-function approach and assessed conse- pLKO cells with the PGE2-AREG combination did not have a quences of expressing a constitutively activated allele of YAP significant impact on cell numbers (Supplementary Fig. S3D). (YAPS127A)inSC4andHSC2l-shNF2 cells. The expression of Taken together, these findings strongly indicate that YAP reg- YAPS127A in both cell lines led to increased expression of COX-2 ulates Nf2-null Schwann cell growth by coordinated regulation and AREG (Supplementary Fig. S5A–S5H and Fig. 6A and B). of AREG and COX-2. Moreover, the expression of YAPS127A also had a significant effect at the cellular level, as evidenced by increases in SC4 (2.5- YAP regulates EGFR signaling in NF2-null Schwann cells fold at 72 hours) and HSC2l (2fold at 72 hours) cell numbers PGE2 is known to promote cell proliferation and inhibit over time, compared with control transfected cells (Fig. 6A and apoptosis through Src-dependent transactivation of EGF receptor B). The effects of YAPS127A expression were dependent on COX- (EGFR) and PI3K/AKT activation downstream (28). We therefore 2, as knocking down COX-2 expression partially rescued the examined the levels and status of EGFR phosphorylation in SC4, growth advantage conferred by expression of YAPS127A (Fig. 6A HEI-193, and HSC2l-shNF2 cells in the absence of YAP. While and B, Supplementary Fig. S5I and S5J). Given the partial rescue total EGFR levels remain unchanged upon YAP knockdown, the by COX-2 knockdown, we next assessed whether the combi- activation of EGFR was greatly reduced as indicated by reduced nation of COX-2 and AREG knockdown would result in a phosphorylation on tyrosine 845 (Fig. 5A and B and Supplemen- greater inhibition. Indeed, combined inhibition led to an tary Fig. S4A and S4B). Similar results were found with knock- almost complete rescue of the growth advantage (Fig. 6C down of COX-2 in HEI-193 cells and by treatment of SC4 cells and D), strongly suggesting that growth advantage conferred with the COX-2 inhibitor celecoxib (Fig. 5B and C). Examination by expression of YAPS127A is mediated through coordinated of the activation status of AKT revealed similar findings. While the regulation of AREG and COX-2. levels of total AKT were not significantly affected by the presence or absence of YAP or COX-2, the phosphorylation at serine 473 Inhibition of COX-2 slows tumor progression in vivo decreased in a YAP- and COX-2–dependent manner (Fig. 5D and Our findings suggest that treatment with a COX-2 inhibitor, Supplementary Fig. S4C). As we previously determined that PGE2 such as celecoxib, could inhibit the growth of Nf2-null schwan- can partially rescue the effects of YAP knockdown at the cellular nomas. To test this hypothesis in vivo, we employed the orthotopic levels (Fig. 4F), we next sought to determine whether the activa- model of NF2 described above. Tumor progression was moni- tion of EGFR and AKT by YAP–COX-2 is mediated by PGE2. tored every other day by bioluminescence imaging, and total flux Indeed, short-term exposure of SC4 cells to PGE2 caused a counts were recorded for each animal. Between 10 and 12 days significant increase in EGFR-Y845 and AKT-S473 phosphoryla- postinjection, animals reached similar flux readings, indicating 6 tion (Fig. 5E). Interestingly, PGE2 treatment also resulted in measurable tumors (flux ¼ 10 counts) and were enrolled ran- elevated YAP levels, suggesting that a feedback loop may exist domly into control (vehicle only, oral, once daily) or celecoxib- between YAP and COX-2. treated cohorts (100 mg/kg, oral, once daily) for a period of 10 Since we observed phosphorylation of EGFR on tyrosine 845 days. Analysis of the flux readings for the animals in the two in response to treatment with PGE2, we next examined the cohorts demonstrated a significantly slower tumor growth rate in possible contribution of Src, which is known to phosphorylate celecoxib-treated mice than in control mice (Fig. 7A–C). Taken EGFR at tyrosine 845 and previously shown to transactivate together, these data demonstrate that inhibition of COX-2 sig- EGFR in response to PGE2 in colorectal cancer cells (29, 30). nificantly impaired Nf2-null Schwann cells growth and tumor Treatment with the Src inhibitor SU6656 alone decreased formation in vivo. phosphorylation of EGFR-Y845, which is perhaps expected, given a basal level of EGFR activity supporting cell survival COX-2 and AREG expression are correlated in NF2-null (Supplementary Fig. S4D). Significantly, pretreatment of cells schwannomas with SU6656 inhibited PGE2-induced EGFR phosphorylation, Our findings suggest that AREG and PTGS2 should be co- implicating Src as a mediator of EGFR activation by PGE2 regulated in NF2-null Schwannoma. To assess this, we examined (Fig. 5F and G, Supplementary Fig. S4E and S4F). Taken the expression of these genes in an existing expression microarray together, these findings strongly indicate that YAP regulates dataset prepared from 31 vestibular schwannomas (31). AREG

Figure 1. YAP is required for NF2-null Schwann cell proliferation and survival. A, SC4 cells were transfected with a control or YAP SMARTpool siRNA mix. B, HEI-193 cells were transfected with control or two independent YAP siRNAs. YAP levels were assessed by Western blotting, and cell numbers were scored at 24, 48, and 72 hours posttransfection. Proliferation of SC4 (C) or HEI-193 (D) cells was assessed by BrdU incorporation at 24, 48, and 72 hours posttransfection. SC4 (E) or HEI-193 (F) cells were treated with the indicated concentrations of verteporﬁn (VP) and cell viability was measured at 24 hours using the CellTiter-Glo assay. G, cells infected with lentiviral control-shRNA or YAP-shRNA were surveyed for markers of apoptosis. The data shown are the mean of three independent experiments, each done in triplicate. Error bars, SD.

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A BC12 P < 0.001 SC4 90 10 SC4 pLKO 80 SC4 pLKO-YAP 5 70 8 60

50 SC4 SC4-Luc pLKOSC4-LucpLKO-YAP 6 40 YAP 30 4 Cells/well × 10

Colonies/well Vinculin 20 2 10 0 0 SC4 pLKO SC4 pLKO-YAP 02448 72 Time (h) D F 2.00E+08 SC4-Luc

Luminescence SC4-Luc pLKO-YAP

1.5 1.50E+08

1.00E+08 P < 0.0001

Flux (photons/s) 5.00E+07

1.0 0 ×107 Time (days): 0 2 4 6 8 10 12 14 16 18 20 E G 6 P < 0.001 0.5 5

2 Radiance 2

(p/sec/cm /sr) ratio (%) Tumor/body 1

Color scale Min = 8.78e5 Max = 1.57e7 0 SC4-Luc SC4-Luc pLKO-YAP

Figure 2. YAP is required for growth of Nf2-null colonies in soft agar and schwannoma in vivo. A, colony formation in soft agar was assessed by plating equal numbers of SC4 pLKO or SC4 pLKO-YAP cells and counting colonies after 14 days. B, SC4-Luc cells were infected with a lentiviral vector expressing control (SC4-Luc pLKO) or YAP shRNA (SC4-Luc pLKO-YAP). YAP levels were assessed by Western blotting 72 hours postinfection. C, cell numbers were assessed by counting at 24, 48, and 72 hours. The data shown in A and C are the mean of three independent experiments, each done in triplicate. Error bars, SD. D and E, representative images from bioluminescence imaging of NOD/SCID mice carrying orthotopic tumors originating from SC4-Luc/pLKO (D) or SC4-Luc/pLKO-YAP (E) at 20 days postsurgery. F, quantitative analysis of flux reading from implanted cohorts. SC4-Luc pLKO control or SC4-Luc pLKO-YAP cells were implanted into the sciatic nerve (day 0). Animals were monitored every 2 days beginning at day 6 (arrow) after implantation, bioluminescence imaging signal was detected starting on day 8. Comparison of the tumor growth trends indicates that the speed of tumor growth in the YAP-knockdown group is significantly slower than control group. Error bars, SEM. G, distribution of tumor/body weight ratios in the SC4-Luc/pLKO or SC4-Luc/pLKO-YAP cohorts. The results of t test with equal variances show the YAP-KD group has significantly lower average tumor weight ratio compared with control group. For the in vivo experiments, n ¼ 15 in each cohort.

and PTGS2 displayed a Pearson correlation coefficient of 0.5784 array data, the levels of AREG and COX-2 protein appear to (P ¼ 0.0007), suggesting that a correlation exists between correlate in the majority of these tumors (Fig. 7G). In a limited expression of both alleles at the transcriptional level (Fig. number of tumors, we were also able to assess the levels of AKT 7D). Interestingly, the expression of PTGS2 also shows a sta- and p-AKT S473, which appear to be consistent between the tistically significant correlation with CYR61 (r ¼ 0.7101, P < samples (Fig. 7G). 0.0001) and CTGF (r ¼ 0.4623, P ¼ 0.0088; Fig. 7E and F), which have been previously characterized as YAP target genes (32). To further confirm the correlation between AREG and Discussion COX-2 at the protein level, we assessed the levels of AREG and While mechanistic details of YAP regulation and down- COX-2 in protein extracts prepared from NF2-null schwanno- stream targets are emerging, relatively little is known about mas. AREG and COX-2 are expressed in the majority of tumors relevance and function of these targets in cancer. YAP has been examined (10 of 16), and in agreement with the expression shown to drive expression of a number of mRNAs and miRNAs

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Figure 3. Targets of YAP-dependent transcription. A, hierarchical clustering of differentially expressed genes between SC4 treated with control or YAP siRNA. Three independent RNA preparations were analyzed. Arrow, YAP. B, list of top 12 genes differently expressed between SC4 treated with control or YAP siRNA, ranked by adjusted P value. C, top 10 enriched canonical pathways on the basis of analysis of differentially expressed genes. D and E, RT-PCR validation of PTGS2 (D) and AREG (E) as YAP-dependent transcriptional targets in SC4 and HEI-193 cells. The data shown are the mean of three independent experiments, each done in triplicate. Error bars, SD. implicated in various aspects of cell proliferation and trans- pathway. However, the role of YAP in NF2-null schwannomas formation including connective tissue growth factor (CTGF), has yet to be established. Our findings demonstrate YAP is thereceptortyrosinekinaseAxl,EGFreceptorligandamphir- required for the survival and proliferation of Nf2-null Schwann egulin (AREG), the cell-cycle regulator cyclin D1, and the cells and tumor formation in vivo. From the broad range of FoxM1 transcription factor (32–35). Previous studies indicate transcripts regulated by YAP, we focused on PTGS2, which codes that YAP can suppress PTEN levels through expression of miR- for COX-2, as it has recently been identified as a direct target of 29 and regulate global miRNA biogenesis through micropro- YAP in a model of pancreatic ductal adenocarcinoma (38). COX-2 cessor activity (36, 37). It is likely that YAP regulates many of expression is typically low in normal tissue but reported as these targets in a cell- and tissue-specific manner, and further elevated in multiple types of cancers including pancreatic, lung, work is required to establish the relevance of these targets in head and neck, prostate, and breast tumors (28). COX-2 is a YAP-dependent tumors. critical enzyme in biosynthesis of prostaglandins, of which PGE2 Merlin, the product of the NF2 tumor suppressor gene, is well- has been identified as playing a major role in promoting tumor established as an upstream regulator of the Hippo signaling growth (39–41).

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A B P < 0.0001 8 7 P < 0.0001 6 5 4 3 AREG (pg/mL) 2 1 0 siRNA: Control YAP AREG

CDE6 12 Control P < 0.05 Control P < 0.05 18 Celecoxib P < 0.05 siRNA 5 siRNA 5 16 5 µ 10 mCOX-2 5 hCOX-2 0 mol/L siRNA #1 14 siRNA #1 30 µmol/L 8 4 12 10 50 µmol/L 6 3 8 4 2 6 4 1 2 × 10 SC4 Cells/well

SC4 Cells/well × 10 SC4 Cells/well 2 HEI-193 Cells/well × 10 HEI-193 Cells/well 0 00 02448 72 0 24 48 72 02448 72 Time (h) Time (h) Time (h)

FGH 12 SC4 pLKO P < 0.05 12 SC4 pLKOP < 0.05 12 SC4 pLKO P < 0.05 5 5 5 SC4 pLKO-YAP SC4 pLKO-YAP 10 SC4 pLKO-YAP 10 10 SC4 pLKO-YAP + SC4 pLKO-YAP + SC4 pLKO-YAP + 8 8 8 PGE2 AREG AREG/PGE2 6 6 6 4 4 4 2 SC4 Cells/well × 10 SC4 Cells/well 2

2 × 10 SC4 Cells/well SC4 Cells/well × 10 SC4 Cells/well 0 0 0 02448 72 0 24 48 72 0 24 48 72 Time (h) Time (h) Time (h)

Figure 4. COX-2 is a downstream effector of YAP and is required for NF2-null Schwann cell growth. A, Western blot analysis of YAP and COX-2 protein levels in SC4 cells transfected with control siRNA, YAP siRNA SMARTpool, or COX-2 siRNA. Vinculin was used as a loading control. B, levels of secreted amphiregulin in culture media of SC4 cells transfected with control, YAP, or AREG SMARTpool siRNA for 48 hours. SC4 (C) or HEI-193 (D) cells were transfected with control or COX-2 siRNA. Cell numbers were scored at 24, 48, and 72 hours posttransfection. E, inhibition of COX-2 with celecoxib inhibits the proliferation of SC4 cells in a dose-dependent manner. PGE2 (F; 1 mmol/L) or AREG (G; 100 ng/mL) partially rescues decreased SC4 cell numbers in response to YAP knockdown. H, combined PGE2 and amphiregulin treatment fully rescues decreased SC4 proliferation in response to YAP knockdown. SC4 cells were stably transfected with control (pLKO) or YAP shRNA (pLKO-YAP)–expressing vectors and treated with PGE2, AREG, or both every 12 hours. Cell proliferation was assessed by counting 24, 48, and 72 hours after initial PGE2 or amphiregulin treatment. The data shown are the mean of three independent experiments, each done in triplicate. Error bars, SD.

Prostaglandins impact tumorigenesis through multiple rectal cancer cell migration and invasion through activation of a mechanisms including modulation of tumor cell proliferation EGFR/AKT signaling axis, through a b-arrestin/Src-mediated intra- and apoptosis, as well as modulation of the tumor microenvi- cellular mechanism (46). ronment and immune responses. Most relevant to our studies are In our studies, we find that PGE2-mediated activation of EGFR/ cell-autonomous functions of PGE2. Specifically, PGE2 promotes AKT signaling promotes cell survival of Merlin-deficient schwan- cell proliferation in colon and lung cancer cells through the Ras- noma cells (Supplementary Fig. S6). This likely reflects a cell- MAPK and GSK3b/b-catenin signaling pathways (42–44), has type–specific difference in the role of this signaling axis between been shown to promote colon tumor cell survival through acti- colorectal cancer and Schwann tumor cells. Indeed, one example vation of a PI3K/AKT/PPARg axis (39, 45), and promotes colo- of different roles undertaken by the same PGE2-activated pathway

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YAP Promotes Tumorigenesis via Activation of EGFR

Figure 5. YAP signals through multiple downstream effectors to drive proliferation and survival. A, Western blot analysis of EGFR levels and activation in SC4 cells transfected with control (pLKO) or YAP shRNA (pLKO-YAP)–expressing vectors. B, analysis of EGFR levels and activation in HEI-193 cells transfected with control, YAP, or COX-2 siRNA. C, analysis of EGFR levels and activation in SC4 cells treated with PGE2 (1 mmol/L, 10 minutes) or celecoxib (20 mmol/L, 8 hours). D, Western blot analysis of COX-2, YAP, and AKT levels and activation in SC4 cells transfected with control or YAP siRNA SMARTpool or COX-2 siRNA. E, Western blot analysis of EGFR and AKT levels and activation in SC4 cells treated with PGE2 (1 mmol/L, 10 minutes). HEI-193 (F) and SC4 (G) cells were treated with PGE2 (1 mmol/L, 10 minutes) in the presence or absence of SU6656 pretreatment (12 hours, 280 nmol/L). Western blot analyses were used to assess EGFR levels and activation. Vinculin or tubulin was used as a loading control, as indicated. DMSO-treated cells were used as negative controls. The experiments shown are representative of three independent experiments.

includes recent studies showing that PGE2, through a PI3K/AKT NF2-associated schwannomas with consistent overexpression signaling cascade, can protect mouse embryonic stem cells from and activation of EGFR family receptors reported (48). It has undergoing apoptosis (47). As far as Src involvement, our studies been suggested that Merlin regulates EGFR internalization and with SU6656 suggest that Src, or another family member, func- signaling in response to cell:cell contact inhibition (49). Our tions as a mediator of the effects of PGE2, and further studies are studies suggest the activation of EGFR is mediated through required to clearly identify the responsible mediator. Interesting- YAP-coordinated expression of AREG and COX-2/PGE2/EGFR ly, the effects of YAP knockdown on Nf2-null Schwann cells were activation (Supplementary Fig. S6). Further studies are required only partially rescued by PGE2 treatment and required supple- to determine whether both mechanisms function in NF2-null mentation of amphiregulin. It has been previously reported that Schwann cells. PGE2-mediated activation of EGFR in colorectal cancer cells is To establish the relevance of YAP coordinated expression of dependent on TGF-a, an EGFR ligand (30). Thus, it is possible that AREG and COX-2 in patients with NF2, we analyzed the expres- in Schwann cells, the YAP-coordinated expression of AREG and sion of COX-2/AREG in NF2-null schwannomas. COX-2 and PTGS2 functions in a similar fashion to what is observed in AREG expression appeared to be correlated both at the mRNA colorectal cancer cells. and protein levels, suggesting they are indeed regulated coordi- Although the Hippo–YAP pathway probably carries out nately. As the number of normal nerve tissue samples in our study additional functions in NF2-null Schwann cells, EGFR-medi- is low, it is difﬁcult to determine whether the fact that COX-2/ ated suppression of apoptosis appears to represent a major AREG do not appear to be co-regulated in these samples is function in promotion of schwannoma tumorigenesis, as sug- signiﬁcant. Interestingly, PTGS2 expression also correlated with gested by rescue of the YAP knockdown phenotype by AREG the expression of CYR61 and CTGF in patient samples, suggestive and PGE2-mediated activation of EGFR/AKT. Indeed, EGFR has of a role for a YAP-regulated transcriptional program in these been previously implicated in the pathogenesis of sporadic and tumors. Clearly more work is needed to establish the extent of

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Figure 6. Promotion of NF2-null Schwann cell growth by YAP is mediated through COX-2 and AREG. The requirement for COX-2 and AREG for YAP-driven cell proliferation was assessed in SC4 (A, top) and HSC2l-shNF2 71-10 cells (B, top) by transfection with either pcDNA control or YAPS127A expression vectors. After 24 hours, cells were reverse transfected with either control or COX-2 pooled siRNA's, seeded, and counted at 24, 48, and 72 hours. Western blotting was used to assess protein levels of YAP and COX-2 (A and B, bottom). Vinculin was used as a loading control. SC4 (C) and HSC2l (D) shNF2 71-10 cells were transfected with either pcDNA control or YAPS127A expression vectors. After 24 hours, cells were reverse transfected with either control or AREG and COX-2 pooled siRNA's, seeded, and counted at 24, 48, and 72 hours to assess changes in cell numbers. Data are the mean of three experiments, with each condition counted in triplicate. Error bars, SD. , P < 0.001.

YAP-regulated pathways involvement and to determine the sig- analysis of patients with vestibular schwannomas demonstrat- nificance of the relative levels of COX-2/AREG expression in ed that aspirin use shows an inverse correlation with tumor tumors versus normal tissue. The finding that AKT appears to be growth (51). Our findings identify a tumor cell-intrinsic mech- activated in all tumor samples examined, compared with normal, anism, through which the effects of daily administration of is not surprising given the central role of PI3K/AKT signaling in celecoxib elicited a pronounced antitumor effect, and suggest tumorigenesis and the fact that multiple upstream effectors feed that long-term treatment with NSAIDs could provide a benefit into this signaling hub. to patients with NF2. A number of reports have indicated that Previous studies describe elevated COX-2 expression in ves- celecoxib has anticancer activity that is COX-2–independent tibular schwannomas, which appear to positively correlate with (52). However, the inhibitory effects we observed on Nf2-null a higher proliferation index (50). Moreover, a retrospective Schwann cell proliferation with direct knockdown of COX-2,

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YAP Promotes Tumorigenesis via Activation of EGFR

Figure 7. Outcomes of celecoxib treatment and analysis of PTGS2/COX-2 and AREG expression in NF2-null schwannomas. Celecoxib treatment inhibits Nf2-null schwannoma tumor growth in vivo. A, SC4-Luc cells were implanted into the sciatic nerve of NOD/SCID mice. Animals were randomized and enrolled into control or treatment cohorts when bioluminescence imaging measurements reached 106 flux counts (day 0). Treatment cohorts were dosed with 100 mg/kg celecoxib daily, whereas control cohorts received vehicle (black arrows). Bioluminescence imaging measurements were recorded every other day for 10 days (red arrows). B, quantitative analysis of flux readings from treated cohorts. Comparison of the tumor growth trends indicates the speed of tumor growth in the celecoxib-treated group is significantly slower than that in control group (P < 0.0002). Error bars, SEM. n ¼ 15 in each cohort. C, representative images from bioluminescence imaging of NOD/ SCID mice carrying orthotopic tumors originating from SC4-Luc cells at the time of enrollment (day 0) or treated with vehicle only or celecoxib at 10 days of treatment (bottom). D–F, assessment of the correlation between PTGS2 and YAP target gene expression. The correlation between PTGS2 and AREG (D), Cyr61 (E), or CTGF (F) mRNA expression in vestibular schwannomas (N ¼ 31). G, Western blot analysis of COX-2, amphiregulin, AKT, and p-AKT levels in protein extracts prepared from NF2-null schwannoma tissue (1–16) and normal nerve tissue (N1–N2). Vinculin was used as a loading control.

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Guerrant et al.

along with pharmacologic inhibition, underscore a significant Writing, review, and/or revision of the manuscript: W. Guerrant, S. Kota, role for COX-2 in NF2-null schwannomas. Future studies A. Stemmer-Rachamimov, J.L. Kissil employing immunocompetent animal models of NF2 will be Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.L. Kissil required to elucidate the effects of COX-2 inhibition on other Study supervision: J.L. Kissil well-documented functions of eicosanoids, including the pro- fl motion of in ammation and effects on the tumor microenvi- Acknowledgments ronment,theroleofPGE2 in immunosuppression and, perhaps The authors thank Dr. David Lim (House Ear Institute) for sharing HEI-193 highly relevant to vestibular schwannomas, proangiogenic cells, Drs. Margaret Wallace and Hua Li (University of Florida) for immortalized activities. human Schwann cells, and Dr. Kirill Martemyanov (Scripps Research Institute) for critical reading of the article. They also thank the Scripps Genomics Core and Disclosure of Potential Conflicts of Interest Gautam Shankar from the Scripps Informatics Core. W. Guerrant is a recipient of the YIA from the Children's Tumor Foundation. No potential conflicts of interest were disclosed.

Authors' Contributions Grant Support Conception and design: W. Guerrant, S. Kota, J.L. Kissil This work was supported by the NIH (NS077952 and CA124495 to J.L. Development of methodology: W. Guerrant, S. Kota, J.L. Kissil Kissil). Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the payment of advertisement provided facilities, etc.): W. Guerrant, S. Kota, S. Troutman, V. Mandati, page charges. This article must therefore be hereby marked in A. Stemmer-Rachamimov, J.L. Kissil accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W. Guerrant, S. Kota, S. Troutman, M. Fallahi, Received April 28, 2015; revised February 23, 2016; accepted March 29, 2016; A. Stemmer-Rachamimov, J.L. Kissil published OnlineFirst May 23, 2016.

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YAP Mediates Tumorigenesis in Neurofibromatosis Type 2 by Promoting Cell Survival and Proliferation through a COX-2−EGFR Signaling Axis

William Guerrant, Smitha Kota, Scott Troutman, et al.

Cancer Res Published OnlineFirst May 23, 2016.

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