LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Published OnlineFirst January 18, 2011; DOI: 10.1158/0008-5472.CAN-10-2164

Cancer Molecular and Cellular Pathobiology Research

Hideki Murakami1, Tetsuya Mizuno1,4, Tetsuo Taniguchi1,4, Makiko Fujii1, Futoshi Ishiguro1,4, Takayuki Fukui2, Shinya Akatsuka6, Yoshitsugu Horio3, Toyoaki Hida3, Yutaka Kondo1, Shinya Toyokuni6, Hirotaka Osada1,5, and Yoshitaka Sekido1,5

Abstract Malignant mesothelioma (MM) is an aggressive neoplasm associated with asbestos exposure. We carried out genome-wide array-based comparative genomic hybridization analysis with 14 MM cell lines. Three cell lines showed overlapping homozygous deletion at chromosome 13q12, which harbored the LATS2 (large tumor suppressor homolog 2) gene. With 6 other MM cell lines and 25 MM tumors, we found 10 inactivating homozygous deletions or mutations of LATS2 among 45 MMs. LATS2 encodes a serine/threonine kinase, a component of the Hippo tumor-suppressive signaling pathway, and we transduced LATS2 in MM cells with its mutation. Transduction of LATS2 inactivated oncoprotein YAP, a transcriptional coactivator, via phosphorylation, and inhibited MM cell growth. We also analyzed LATS2 immunohistochemically and found that 13 of 45 MM tumors had low expression of LATS2. Because NF2 is genetically mutated in 40% to 50% of MM, our data indicate that Hippo pathway dysregulation is frequent in MM cells with inactivation of LATS2 or an upstream regulator of this pathway, Merlin, which is encoded by NF2. Thus, our results suggest that the inactivation of LATS2 is one of the key mechanisms for constitutive activation of YAP, which induces deregulation of MM cell proliferation. Cancer Res; 71(3); 1–9. 2011 AACR.

Introduction shown to enhance mesothelioma development after asbestos exposure (16, 17). Mesothelioma also develops with a high Malignant mesothelioma (MM) is an aggressive neoplasm incidence in Nf2;Arf conditional knockout mice (18). However, associated with asbestos (1–4). Because MM is usually it remains unclear whether MM tumors without an NF2 diagnosed at advanced stages and is largely unresponsive mutation express functional Merlin or the tumor-suppressive to conventional therapy, the prognosis of patients with MM activity of Merlin is inactivated by other mechanisms. In this is very poor (5, 6). MM shows frequent mutation of p16INK4a/ regard, possible involvement of the increased expression of p14ARF and NF2 (neurofibromatosis type 2) tumor suppressor CPI-17, a regulator of Merlin, or the upregulation of microRNA genes (TSG) and recent comprehensive analyses have that might target NF2 has been suggested (19, 20). identified other candidate cancer-associated genes respon- The mammalian Hippo cascade, which was initially identi- sible for MM development, progression, and poor outcome fied via genetic studies in Drosophila, is one of the possible (7–10). downstream signaling cascades of Merlin and Expanded (21– The NF2 gene, which encodes Merlin, is inactivated in 40% 25). This pathway controls tissue growth by inhibiting cell to 50% of MMs (11–13). Transduction of NF2 into MM cells proliferation and by promoting apoptosis. The components of was shown to inhibit cell proliferation and invasiveness of MM this pathway include SAV1 (also called WW45), MST (Droso- cells (14, 15). Mouse models with nf2 allele loss have been phila Hippo), and LATS family members, which ultimately phosphorylate and inactivate the YAP transcription coactivator. YAP, a candidate oncogene, was shown to be amplified in Authors' Affiliations: 1Division of Molecular Oncology, Aichi Cancer human cancers (26, 27). We previously reported amplification Center Research Institute; Departments of 2Thoracic Surgery and 3Thoracic Oncology, Aichi Cancer Center Hospital; Departments of of the chromosomal 11q22 region including YAP in a subset of 4Cardio-Thoracic Surgery and 5Cancer Genetics, Program in Function MM specimens and a positive role of YAP in MM cell pro- Construction Medicine, and 6Pathology and Biological Responses, liferation (28). Nagoya University Graduate School of Medicine, Nagoya, Japan In this study, we carried out array-based comparative Note: Supplementary data for this article are available at Cancer Research genomic hybridization (CGH) and sequencing analyses and Online (http://cancerres.aacrjournals.org/). found that 10 of 45 MMs had an inactivating homozygous Corresponding Author: Yoshitaka Sekido, Division of Molecular Oncol- ogy, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, deletion or mutation of LATS2. Furthermore, we showed that Nagoya, Aichi 464-8681, Japan. Phone: 81-52-764-2983; Fax: 81-52-764- transduction of LATS2 induced phosphorylation of YAP and 2993. E-mail: [email protected] inhibited MM cell growth. Our results suggest that the Merlin- doi: 10.1158/0008-5472.CAN-10-2164 Hippo pathway is frequently inactivated in MM cells and that 2011 American Association for Cancer Research. LATS2 is a TSG of MM.

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Materials and Methods penetrance per feature ¼ 0). At a minimum, 2 contiguous suprathreshold probes were required to define a change. To Cell lines and primary specimens of malignant find an obvious homozygous deletion in cell line DNA, aber- mesothelioma rant regions with a signal log2 ratio of less than 1.6 were Fourteen Japanese MPM (malignant pleural mesothelioma) searched. Genomic positions were based on the UCSC cell lines, including ACC-MESO-1, -4, Y-MESO-8D, -9, -12, -14, March 2006 human reference sequence (hg18; NCBI build -21, -22, -25, -26B, -27, -28, -29, and -30, were established in our 36.1 reference sequence). The accession number of array laboratory as reported previously and described elsewhere, CGH analysis data to Gene Expression Omnibus is and the cells at 10 to 15 passages were used for assays (29, 30). GSE22237. For tumor tissue DNA, regions of homozygous Four MPM cell lines, including NCI-H28, NCI-H2052, NCI- deletion or one allelic loss of the LATS2 locus were defined < < H2373, and MSTO-211H, and one immortalized mesothelial as log2 ratio 1.0 or 1.0 0.4 for at least 3 consecutive cell line, MeT-5A, were purchased from the American Type probes, respectively. Culture Collection (ATCC) and cells at 3 to 5 passages were used after receiving from ATCC. NCI-H290 and NCI-H2452 Mutation analysis were the kind gifts of Dr. Adi F. Gazdar. All MPM cell lines were Mutation analysis of all coding exons of the LATS2 and SAV1 cultured in RPMI 1640 medium supplemented with 10% fetal and NF2 genes was carried out by direct sequencing after PCR calf serum (FCS) and 1 antibiotic-antimycotic (Invitrogen) amplification of genomic DNA. The primer sets of LATS2 are at 37 C in a humidified incubator with 5% CO2. MeT-5A was described in Supplementary Materials and Methods. The cultured according to ATCC instructions. MM tissue samples primer sets of NF2 were described previously (11, 29), and from patients treated at Aichi Cancer Center Hospital, Nagoya sequences of the primer sets of SAV1 are available upon University Hospital, Japanese Red Cross Nagoya First Hospital, request. Toyota Kosei Hospital, and Kasugai City Hospital were obtained according to the Institutional Review Board– Antibodies and reagents approved protocol for each and the written informed consent Rabbit anti-LATS2 antibody (NB200-199) for Western blot from each patient. The human mesothelioma tissue array with analysis was purchased from Novus Biologicals, and mouse 19 MM samples was also used (US Biomax Inc.). anti-YAP (clone 2F12, H00010413-M01) and anti-SAV1 (clone 3B2, H00060485-M02) antibodies were from Abnova. Rabbit Preparation of DNA and RNA anti-LATS2 antibody (ab70565) for immunohistochemistry Genomic DNA was extracted using a standard phenol– and rabbit anti-YAP antibody (EP1674Y) were purchased from chloroform method (31). Total RNA was prepared using Abcam, and anti-NF2 (1C4, #9169) and anti-phospho-YAP RNeasy Plus RNA extraction kit (Qiagen K.K.) according to (S127; #4911) antibodies were from Cell Signaling Technology. the manufacturer's protocol. Random-primed, first-strand Anti-b-actin (clone AC74) and anti-Flag (M2) antibodies were cDNA was synthesized from 3 mg of total RNA, using Super- from Sigma, and anti-V5 antibody was from Invitrogen. Rabbit script II, according to the manufacturer's instructions (Invi- anti-b-catenin (SC-7199) was from Santa Cruz Biotechnology. trogen). Plasmid and lentiviral vector Oligonucleotide array CGH analysis The cDNA fragments of wild-type or mutant LATS2 were All microarrays used were Agilent 244K whole human amplified by PCR, using PrimeSTAR Max DNA polymerase genome microarrays, with an average distance of 6.4 kb (Takara Bio), and introduced into the pFLAG-CMV2 expres- between each probe (array G4411B sourced from the NCBI sion vector (Sigma) with an infusion cloning system (Clon- genome Build 36; Agilent Technologies). Comparison genomic tech), thereby fusing these cDNAs with the FLAG sequence. DNA was obtained commercially (Promega) and matched for The sequences of all constructs were confirmed. To generate sex. The methods for labeling, hybridization, and scanning LATS2-expressing lentiviral vector, cDNA coding for the using a G2505B Agilent DNA microarray scanner (Agilent human LATS2 tagged with FLAG was amplified by PCR and Technologies) were conducted according to the man- cloned in the pLL3.7 lentiviral vector. NF2 expression vectors ufacturer's protocol. The scanned TIFF image data were were described previously (28). RNA interference vectors to processed with Agilent Feature Extraction software (version generate lentiviruses that transcribe short hairpin (sh)-RNA 9.5.3.1) by the CGH-v4_95_Feb07 protocol (Agilent Technol- were prepared as described previously (32). sh-LATS2-RNA ogies). Extracted data were analyzed with Agilent DNA Ana- interference vector (sh-LATS2) contained a target sequence of lytics 4.0 software (version 4.0.81; Agilent Technologies), and the hairpin loop of LATS2 (50-GGACCTCACTGCATTAAA-30). the Aberration Detection Method 2 (ADM-2) algorithm was A control shRNA vector for luciferase (Sh-Luc), which con- used to identify contiguous genomic regions that corre- tained a target sequence for luciferase (50-CGTACGCGGAA- sponded to chromosomal aberrations. The following para- TACTTCGA-30), was described previously (32). meters were used in this analysis: threshold of ADM-2: 5.0; centralization: ON (threshold: 5.0, bin size: 10); aberration Cell proliferation assays filters: ON (minimum number of probes in region ¼ 2 and A total of 1.0 104 and 2.0 105 cells were seeded onto flat- minimum absolute average log ratio for region ¼ 1.6 bottomed 24- and 12-well plates, respectively. Cells were trans- and maximum number of aberrant regions ¼ 10,000 and % duced with lentiviral vectors at the multiplicity of infection of 5,

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incubated for an additional 6 hours, and then changed with that the homozygous deletion region of NCI-H2052 was RPMI 1640 medium with 5% or 1% FCS. Cells were incubated for different from that of the others (data not shown). We further an additional 90 hours. Each viral transduction was applied to found short PCR products in 2 cell lines and confirmed that triplicate wells for cells. Cell numbers were counted under a light MSTO-211H had a 42-bp deletion in exon 5 and that Y-MESO- microscope every 24 hours. Calorimetric assays were carried out 30 had a 125-bp deletion, including 14-bp deletion of exon 6. with the addition of 100 mL of TetraColor One (Seikagaku), We also found a nonsense mutation, Y649X, in Y-MESO-26B. containing 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- Figure 2B summarizes 7 genetic mutations of LATS2 in 20 MM (2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, and 1- cell lines (Fig. 2B). Because the original tumors were available methoxy-5-methylphenazinium methylsulfate as electron car- for 2 cell lines with LATS2 mutation, we analyzed them. In case rier, in each well and then incubating at 37Cfor1hour. of Y-MESO-30, we detected the same short fragment of exon 6 Absorbance was read at 450 nm with a multiplate reader. in the primary tumor but not in lymphocytes (Fig. 2C). This Growth inhibition was expressed as a mean ratio of absorbance result indicated that the 125-bp deletion was somatic, which reading from treated versus untreated cells. caused an aberrant transcript of LATS2 (Fig. 2D). We also confirmed the nonsense mutation in the primary tumor of Y- Immunofluorescent microscopic analysis MESO-26B, using a mutation-specific primer set (data not Cells were fixed in 4% paraformaldehyde for 20 minutes on shown). ice, followed by permeabilization with PBS containing 0.3% To validate LATS2 mutation in primary MM tumors, we Triton X-100 for 3 minutes. Slides were blocked in PBS analyzed another cohort of 25 primary tumors with array CGH containing 3% goat normal serum for 20 minutes at room and/or sequencing analyses. We found that 2 tumors had temperature (RT). Samples were stained with primary anti- significant loss (their log2 ratio values were 2.5 and 1.6, bodies (mouse anti-YAP antibody, 1 mg/mL; anti-b-catenin respectively), which indicated homozygous deletion, and 1 antibody, 1 mg/mL) for overnight at 4C, followed by incuba- had a somatic mutation at the intron 6–exon 7 boundary tion with Alexa Fluor 488- or 564-conjugated secondary anti- (c2676-3C>A), which caused an aberrant transcript (data not body for 30 minutes at RT. Nuclear staining was carried out shown). Thus, we considered that 3 (12%) of 25 had a genetic with DAPI after incubation with secondary antibodies. The alteration of LATS2 to inactivate. Furthermore, 5 tumors had a slides were mounted with PermaFluor Mounting Medium loss of LATS2 with array CGH analysis, which also suggested a (Thermo). Microscopic observation was carried out using possibility of LATS2 inactivation in the other allele, though we an Carl Zeiss LSM510 confocal laser scanning system at could not determine any of them as a homozygous deletion 63 magnification. because of the contamination of normal cell populations in Additional materials and methods are described in Supple- the tumors (data not shown). mentary Materials and Methods. SAV1 is homozygously deleted in one MM cell line Results SAV1 was reported to be deleted in renal cancer cell lines (33). Array CGH analysis detected a homozygous deletion at Homozygous deletions at 13q12.11 region in MM cells chromosome 14q22 in Y-MESO-28, and PCR analysis con- We carried out array CGH analysis with 14 MM cell lines. firmed complete deletion of exons 1 and 2 of SAV1 (data As expected, we detected homozygous deletions at 9p21 and not shown). However, sequencing analysis of SAV1 did not 22q12 in multiple cell lines, which harbor p16Ink4a/p14Arf detect mutations in other cell lines. (CDKN2A/2B)andNF2, respectively (data not shown). Three cell lines showed homozygous deletion at 13q12.11, and the Comparison of inactivation status of Merlin, LATS2, deletion regions in the cell lines Y-MESO-14 and Y-MESO-27 and SAV1 in MM cell lines were overlapped and the one in Y-MESO-21 was located 20 We analyzed the mutation and expression status of NF2 and to 30 kb away from them (Fig. 1A). FISH analysis also compared the inactivating status among Merlin, SAV1, and confirmed a deletion in Y-MESO-14 cell line (Fig. 1B). LATS2 (Fig. 3 and Table 1). Among 20 MM cell lines, 15 (75%) Because a single gene, LATS2,waslocatedinthisdeletion showed inactivation for 1 of the 3 genes, with 3 (Y-MESO-14, Y- region and homozygous deletions of all 3 cell lines disrupted MESO-26B, and NCI-H2052) having inactivation of 2 genes. this gene, we considered LATS2 to be a strong candidate for a Because 2 MM cell lines (Y-MESO-28 and Y-MESO-8D) with- TSG of MM. Meanwhile, we did not detect any deletion at out NF2 mutation did not express Merlin, 16 (80%) cell lines chromosome 6q25.1, which harbors another LATS homo- were considered to be inactivated in the Merlin-Hippo path- logue, LATS1 (data not shown). way.

LATS2 mutations in MM cells Dysregulation of Merlin-Hippo signaling in MM cells We carried out mutational analysis of LATS2 with the 14 cell When cells grow and become confluent, cell surface signals lines with other 6 MM cell lines. Genomic PCR analysis transmit via the Hippo signaling pathway and activated LATS confirmed the homozygous deletions in the 3 cell lines, with phosphorylates YAP, a transcriptional coactivator (25). The compatible deletion patterns of the array CGH analysis phosphorylated YAP (inactivated form as a transcriptional (Fig. 2A). Because we detected another homozygous deletion coactivator) is translocated to the cytoplasm, which results in NCI-H2052, we carried out array CGH analysis and found in cell contact inhibition (25). To determine whether the

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Figure 1. Deletion of chromosome 13q12.11 region in MM cells. A, 3 cell lines showed a high-level loss with multiple probes, indicating a homozygous deletion. Y-MESO-14 and Y-MESO-27 had a deletion of exon 3–8 and Y-MESO-21 had a deletion of exon 1–2ofLATS2. B, FISH analysis detected loss of the LATS2 region (RP11-23H13 probe, green signal) but not 13q telomeric region (RP11-1148G1, red signal) in Y-MESO-14 cell line.

inactivated Merlin-Hippo signaling pathway in MM cells could duced (Supplementary Fig. 1). We found that LATS2 knock- be reactivated, we transduced NF2 or LATS2 expression con- down significantly blocked phosphorylation of YAP with NF2 structs. NF2 transduction in NCI-H290 cells with NF2 deletion transduction, suggesting that LATS2 is a crucial mediator of induced YAP phosphorylation (Fig. 4A). In contrast, NF2 Merlin-Hippo signaling and that LATS1 might have only a transduction in Y-MESO-14 that has both NF2 and LATS2 minor role, if any, in the phosphorylation of YAP in MM cells. mutations did not induce YAP phosphorylation, suggesting We also confirmed that transduction of the wild-type LATS2 that LATS2 was necessary to transmit a growth inhibitory induced phosphorylation of YAP in MM cells with LATS2 signal from Merlin to YAP (Fig. 4A). Furthermore, we carried deletion (Fig. 4B). However, the mutant LATS2 (LATS2-delEx6) out a knockdown experiment with a shRNA expression vector construct, which deleted exon 6, did not induce YAP phos- of LATS2 and tested whether YAP phosphorylation in NCI- phorylation, indicating that the mutant detected in the H290 cells could be blocked when wild-type NF2 was trans- Y-MESO-30 cell line was functionally inactive.

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Figure 2. Genetic inactivations of LATS2 in MM cells. A, genomic PCR analysis detected homozygous deletion in Y-MESO-14, -21, -27, and NCI-H2052, and shorter fragments in Y-MESO-30 and MSTO-211H cell lines (arrows). B, a diagram of 7 LATS2 inactivating mutations in MM cell lines. C, genomic PCR analysis detected a shorter product by 125 bp in the Y-MESO-30 cell line and its corresponding tumor but not in lymphocytes. This deletion disrupted the donor site of the exon 6 boundary (14 bp in exon 6 and 111 bp in intron 6). D, RT-PCR analysis covering exon 5–7 detected a shorter fragment skipping exon 6, which caused the deletion of 61 amino acids coded by 183 nucleotides of exon 6.

LATS2 acts as a growth suppressor in MM cells formation and Transwell migration assays and found that To determine whether the regulation of YAP by cell density LATS2 transduction in MM cell lines with LATS2 mutation was abrogated in MM cells, we then analyzed the change of inhibited both activities in these cell lines (Fig. 5B and C and cellular localization of YAP with immunocytochemistry. All 3 Supplementary Fig. 2B). MM cell lines with LATS2 mutation showed nuclear accumu- Finally, we carried out a knockdown experiment of LATS2 lation of YAP even at high cell density, whereas MeT-5A, in MeT-5A cells to determine whether silencing of LATS2 immortalized, nonmalignant mesothelial cells, showed promotes cell growth of nonmalignant mesothelial cells. nuclear accumulation at low cell density but presented cyto- LATS2 knockdown significantly decreased YAP phosphoryla- plasmic translocation at high cell density (Fig. 4C). As tion status and slightly increased YAP protein level (Supple- expected, Western blot analysis showed that the subcellular mentary Fig. 3A). We found that silencing of LATS2 increased localization change of YAP in MeT-5A according to high cell the cell proliferation of MeT-5A cells under low serum con- density was accompanied with a significant increase in phos- dition (Supplementary Fig. 3B and C), but the colony forma- phorylated YAP (Fig. 4D). In contrast, the basal levels of YAP tion in soft agar was not enhanced (data not shown). These phosphorylation in MM cells were low and there was only a results suggested that LATS2 was involved in the regulation of modest increase in YAP phosphorylation levels (Fig. 4D). cell proliferation of nonmalignant mesothelial cells as well as To determine whether LATS2 has a growth-suppressive MM cells. activity, we transduced the both wild-type and mutant LATS2 constructs in MM cells. Transduction of the wild-type, but not Immunohistochemical analysis of LATS2 and YAP in the mutant, LATS2 inhibited cell proliferation of MM cells with primary MMs LATS2 mutation, indicating that LATS2 acts as a growth To determine whether immunostaining can be useful to suppressor in MM cells in vitro (Fig. 5A and Supplementary detect LATS2 inactivation status in MMs, we carried out Fig. 2A). We also carried out anchorage-independent colony immunohistochemical analysis with anti-LATS2 antibody

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Figure 3. Western blot analysis of Merlin, SAV1, and LATS2. LATS2 protein was undetectable in 5 cell lines harboring a homozygous deletion or a premature termination, although faint nonspecific bands with slower mobility than the wild-type LATS2 were observed (indicated by asterisks). Aberrant short LATS2 proteins were detected in MSTO- 211H and Y-MESO-30 (indicated by "S"). Expression of b-actin was used as the control.

(Fig. 6). The 2 MM tumors with homozygous deletion suggesting that 13 (29%) of 45 primary MMs had down- detected by array CGH analysis showed negative or only regulation of LATS2. weak staining of LATS2, suggesting that weak (1þ)signals We also carried out immunohistochemical analysis to deter- might be caused by nonspecific staining. Among 45 cases, 2 mine how frequently primary MMs show YAP activation (Fig. 6). showed negative and 11 showed weak staining of LATS2, Among 45 cases, 36 showed positive staining for YAP and 33 whereas 32 had moderate or strong staining of LATS2, tumors showed stronger or equal staining of YAP in the nucleus

Table 1. Inactivation of NF2, LATS2, and SAV1 in MM cell lines

Cell line NF2a LATS2 SAV1

NCI-H290 HD þþ NCI-H2373 HD þþ ACC-MESO-1 Q389X þþ Y-MESO-9 NM_000268:c.527_528del2 þþ Y-MESO-12 HD þþ Y-MESO-22 HD þþ Y-MESO-25 NM_000268:c.532_571del40 þþ Y-MESO-14 Q196X HD þ Y-MESO-26B HD Y649X þ NCI-H2052 R341X HD þ Y-MESO-21 þ HD þ Y-MESO-27 þ HD þ Y-MESO-30 þ NM_014572:c.2652_2665þ111del125 þ MSTO-211H þ NM_014572:c.2355_2396del42 þ Y-MESO-28 b þ HD Y-MESO-8D b þþ NCI-H28 þþ þ NCI-H2452 þþ þ ACC-MESO-4 þþ þ Y-MESO-29 þþ þ

Abbreviation: HD, homozygous deletion. aMutation status of NF2 in 9 cell lines (ACC-MESO-1, -4, Y-MESO-8D, NCI-H28, -H290, -H2052, -H2373, -H2452, and MSTO-211H) was previously described (11, 29, 30), and the one in the other 11 cell lines was analyzed in the present study. bSilenced expression.

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A BC

D D

Figure 4. Inactivation of the Merlin-Hippo signaling pathway in MM cells. A, wild-type NF2 (NF2-WT), but not truncated NF2 (NF2-mt), induced phosphorylation of YAP in NCI-H290 cells with NF2 homozygous deletion (HD). In contrast, wild-type NF2 did not induce phosphorylation of YAP in Y-MESO-14 cells with both NF2 mutation and LATS2 HD. B, wild-type LATS2, but not mutant LATS2 with exon 6-deletion (LATS2-delEx6), induced phosphorylation of YAP in Y-MESO-27 cells with LATS2 HD, indicating that this aberrant form detected in Y-MESO-30 was kinase dead. C, MeT-5A showed YAP translocation in the cytoplasm at high cell density, whereas YAP in MM cells remained in the nucleus at high cell density. D, YAP phosphorylation according to the increasing cell density was induced in MeT-5A, whereas the basal levels of phospho-YAP (pYAP) were low at low cell density in the 3 MM cell lines. Only a modest increase in YAP phosphorylation was observed according to the higher cell density. S, short exposure; L, long exposure.

than in cytoplasm, indicating constitutive YAP activation in into MM cells with LATS2 mutation induced phosphorylation more than 70% of primary MMs (Supplementary Table 1). of YAP, which resulted in suppression of MM cell proliferation We finally studied the relation of LATS2 expression with and anchorage-independent growth. Our study indicates that YAP activation status (Supplementary Table 2). Among 13 LATS2 may be a TSG in MM cells. tumors with negative or weak LATS2 expression, 11 had Merlin is a membrane–cytoskeleton-associated protein stronger or equal staining of YAP in the nucleus, suggesting with an FERM (Four-point-one, Ezrin, Radixin, and Moesin) that negative or weak LATS2 may be an indicator of YAP domain, and is known to interact with 34 proteins, including activation. CD44, ERM (ezrin radixin moesin) proteins, and PAK1 (p21- activated kinase 1; ref. 34). The prevalence of NF2 mutations in Discussion sporadic tumors, especially schwannomas, meningiomas, and MMs, suggest that Merlin has a relatively broad tumor sup- In the present study, we showed that LATS2 was genetically pressor function (35, 36). Merlin and the ERM proteins have inactivated in 7 of 20 MM cell lines and 3 of 25 primary tumors. been suggested to function to both stabilize the membrane– We found that MM cells with LATS2 mutation showed con- cytoskeleton interface and to organize the distribution of, and stitutive activation of YAP with underphosphorylation, regard- signaling by, membrane receptors (37). Merlin exerts inhibi- less of high cell density, whereas YAP in nonmalignant tory effects on multiple mitogenic signaling pathways such as mesothelial cells was phosphorylated and inactivated at high RAS-ERK, PI3K-AKT, and mTOR. A recent study also indicated cell density. We further showed that transduction of LATS2 that a closed, growth-inhibitory form of Merlin accumulates in

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Figure 5. LATS2 acts as a TSG in MM cells. A, inhibition of cell proliferation of Y-MESO-27 and Y- MESO-30 cells by re-expression of LATS2. Cells were transduced with wild-type LATS2 (LATS2- WT), mutant LATS2 (LATS2- delEx6), or GFP viruses (GFP), or BCuninfected (Cont.). Cell numbers were counted every 24 hours. Points, mean; bars, SD. B, re- expression of LATS2-WT in MM cell lines suppressed anchorage- independent colony formation. Representative results of the Y- MESO-27 and Y-MESO-30 cell lines are shown (top) with higher magnifications of their representative colonies (bottom). C, the numbers of colonies in the triplicate experiments are presented. Columns, mean; bars, SD.

the nucleus, binds to the E3 ubiquitin ligase CRL4DCAF1, and homozygous deletion or nonsense mutation was not suppresses its activity (38). In addition to these pathways, the reported in these malignancies. Hippo pathway is thought to be one of the downstream Why only 40% to 50% of MMs have NF2 mutation and the signaling pathways of Merlin, which is regulated via signaling rest do not has been a long-standing enigma. The represen- with cell–cell adhesion, cell–cellular matrix, or other cell tative hypotheses for them are that MM tumors without an membrane receptors with binding of extracellular ligands (21). NF2 mutation may not express functional Merlin, or that the Dysregulation of the Hippo pathway causes an increase in other molecules of Merlin-associated signaling cascades are organ size both in Drosophila and in mammalians (22). The altered. Supporting the former hypothesis, one study indicated recent findings indicated that a variety of human malig- that Merlin was phosphorylated on Ser518 if present and nancies, such as homozygous deletion of SAV1 in renal functionally inactivated in MM cells with elevated CPI-17, a cancer cell lines (33) and hypermethylation of MST in soft cellular inhibitor of myosin phosphatase MYPT1-PP1d (19), tissue sarcoma (39), have alterations in each component. and the other showed that upregulation of microRNAs, such Overexpression of YAP was reported in hepatocellular car- as hsa-miR-885-3p, might target NF2 (20). Meanwhile, our data cinomas (40) and colonic and lung adenocarcinomas (41). In may explain the latter hypothesis, indicating that one of the our previous study, we also reported YAP amplification in a major downstream pathways of Merlin can be inactivated subset of MM cells (28). Regarding LATS2, downregulation of with an LATS2 or SAV1 mutation. We think of the idea that LATS2 was reported to be correlated with poor prognosis of LATS2 is a TSG of MM is supported by the evidence that the leukemia (42) and missense mutation was also reported in mutation frequency of LATS was in 22% (10 of 45 MMs lung cancer (43). However, null status of LATS2 such as by including 20 cell lines and 25 primary tumors), and that the

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Figure 6. Immunohistochemical analyses of LATS2 and YAP in 45 primary MMs. Representative cases of LATS2 and YAP staining are presented. Case KD1074 with LATS2 homozygous deletion and case KD1067 with LATS2 deletion showed negative (0) and weak (1þ) LATS2 staining, respectively. Both cases showed stronger staining of YAP in the nucleus. Meanwhile, cases KD1072 and KD1077 without LATS2 deletion showed moderate (2þ) and strong (3þ) LATS2 staining, respectively. N, nucleus; C, cytoplasm.

characteristics of inactivation mechanisms were direct and Merlin has been clearly shown to inhibit mTORC1 pathway in robust by a homozygous deletion, small deletion, or nonsense MM cells, with Merlin-negative MM cells displaying unregu- mutation. To our knowledge, our study is the first to show lated mTORC1 signaling and also an enhanced growth- such frequent genetic inactivation of the LATS2 gene in any inhibitory effect of rapamycin, an mTORC1 inhibitor (44, human malignancy. 45). Thus, growth suppression in Y-MESO-14 cells was likely Interestingly, several MM cell lines show inactivation of to be induced via such signaling cascades but not via the both NF2 and LATS2. This is in contrast to our hypothesis that Hippo signaling. This suggests another possibility that the the functional link between the Merlin and Hippo pathway main roles of Merlin for tumor-suppressive activity in MM was direct and that inactivation of each gene might be cells reside outside the Hippo pathway regulation, that the sufficient for the inactivation of the Merlin-Hippo cascade functional link between the Merlin and Hippo pathway in MM in MM cells. Indeed, although underphosphorylated, active cells is not as direct as expected, and that the simultaneous YAP in the Y-MESO-14 cell line that had both gene inactiva- inactivation of Merlin and Hippo pathway inactivation may tions was not downregulated by phosphorylation when the merely enhance MM cell growth. Furthermore, some MM wild-type NF2 gene was transduced (Fig. 4A), the cell growth of cell lines with NF2 mutation but not LATS2 mutation also this cell line was suppressed (data not shown). In this regard, showed significant phosphorylation levels of YAP, especially at

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Murakami et al.

confluence, suggesting that the Hippo pathway in MM cells nonmalignant mesothelial cells. Our result may also suggest can also be activated in a Merlin-independent manner (data that inhibition of activated YAP or transcription factors may not shown). Thus, more detailed mechanisms of the tumor- serve to develop a more effective target therapy for patients suppressive pathways in which Merlin and LATS2 are involved with MM in the future. must be elucidated in future studies. The mutation frequency of LATS2 in MM cell lines was higher than the one in primary tumors. Because most Disclosure of Potential Conflicts of Interest primary MM tumors contain abundant normal cells, the No potential conflicts of interest were disclosed. sensitivities of detection of allelic loss or point mutation in primary tumors are expected to be lower than those in cell Acknowledgments lines. In this regard, among 5 primary tumors that we evaluated to have at least an allelic loss, there might be We thank Dr. Adi F. Gazdar for the cell lines and Ms. Mari Kizuki and Ms. cases of homozygous deletion. Indeed, 3 of 4 tumors with an Mika Yamamoto for their excellent technical assistance. allelic loss showed the weak intensity of LATS2 staining comparable with a tumor with homozygous deletion, which Grant Support suggested that both alleles of LATS2 might be inactivated in these tumors. Thus, although the mutation frequency of 12% This work was supported in part by a Special Coordination Fund for for LATS2 in primary tumors was low compared with that of Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H18-1-3-3-1), grants-in-aid for Scien- 35% in cell lines, we thought that one reason for this was the tific Research (B) from Japan Society for the Promotion of Science (18390245, lower sensitivity of mutation detection. However, we could 22300338), grant-in-aid for Third-Term Comprehensive Control Research for not exclude another possibility, namely, that the difference Cancer from the Ministry of Health, Labor and Welfare of Japan, and the Takeda Science Foundation. in mutation frequencies was due to selection pressure dur- The costs of publication of this article were defrayed in part by the ing establishment of cell lines. payment of page charges. This article must therefore be hereby marked In conclusion, we showed that the tumor-suppressive advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Hippo signaling pathway can be inactivated by LATS2 mutation in MM cells and that LATS2 may play a critical role in Received June 14, 2010; revised November 30, 2010; accepted November 30, regulating cell proliferation and/or survival in MM cells and 2010; published OnlineFirst January 18, 2011.

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LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Hideki Murakami, Tetsuya Mizuno, Tetsuo Taniguchi, et al.

Cancer Res Published OnlineFirst January 18, 2011.

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