Oncogene (2015), 1–13 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE

LPP inhibits collective cell migration during lung cancer dissemination S Kuriyama1, M Yoshida2, S Yano3, N Aiba1, T Kohno4, Y Minamiya5, A Goto2 and M Tanaka1 Lipoma preferred partner (LPP) is a LIM domain protein, which has multiple functions as an actin-binding protein and a transcriptional coactivator, and it has been suggested that LPP has some roles in cell migration or invasion, however, its role in cancer cells remains to be elucidated. Here, we showed that LPP degraded N-cadherin in lung cancer, PC14PE6 cells via regulating the expression of matrix metalloproteinase 15 (MMP-15), and loss-of-LPP increases collective cell migration (CCM) and dissemination consequently. Knockdown of LPP and its functional partner, Etv5, markedly restores the full-length N-cadherin and increases cell–cell adhesion. We investigated the common target of LPP and Etv5, and found that MMP-15 is transcribed as their direct transcriptional target. Furthermore, MMP-15 could directly digest the N-cadherin extracellular domain. LPP knockdown in PC14PE6 cells increases N-cadherin-dependent CCM in the three-dimensional collagen gel invasion assays, and promoted the dissemination of cancer cells when they were orthotopically implanted in nude mice. Immunohistochemistry of lung adenocarcinoma specimens revealed the heterogeneity of LPP intensity and complementary expression of LPP and N-cadherin in the primary tumors. These findings suggest that loss-of-LPP, Etv5 or MMP-15 can be a prognostic marker of increasing malignancy. Oncogene advance online publication, 1 June 2015; doi:10.1038/onc.2015.155

INTRODUCTION Lipoma preferred partner (LPP) gene LPP locates on chromosome 3q27-q28, and is characterized by a consistently rearranged chromosome segment of high mobility group gene allele at 12q15 in lipoma.1 LPP localizes to focal adhesions (FAs),2 and the N-terminal domain of LPP can interact with alpha-actinin3 and Ena/VASP,2,4 which are involved in actin cytoskeleton. The C-terminal half of LPP is well-conserved LIM domains interacting with PEA3/Ets variant (Etv) genes.5–8 Etv5 in association with LPP regulates epithelial-to-mesenchymal transition (EMT) in endometrial carcinomas.9 These suggest that LPP has multiple partners and reflects its various functions in a context-dependent manner. LPP has similar LIM domains to TRIP6, which binds to the LPAR2 receptor.10 During previous study in embryonic development, we found that N-cadherin is precisely regulated for proper collective cell migration (CCM) downstream of LPAR2,11 and LPP also binds to LPAR2 (data not shown), which is tempting to investigate if LPP is involved in N-cadherin-dependent cell adhesion in cancer. N-cadherin functions as a cell surface receptor for neural cell adhesions.12 The loss of E-cadherin and de novo expression of N-cadherin are often observed in cancer EMT,13 which promotes multiple processes associated with metastasis in breast cancer,14,15 melanoma16,17 or prostate cancer.18 Conversely, N-cadherin overexpression in the rat glioma C6 cells reduces invasiveness,19 and the reduction of N-cadherin expression is correlated with high-grade gliomas and the knockdown of N-cadherin in lower-grade glioma increases speed of cell migration but reduced directed migrations.20 Thus, we cannot

simply predict how cancer cells behave from the level of N-cadherin expression. Moreover, in the development, N-cadherin is required for the directional CCM via SDF1-CXCR4 interaction,21 conversely, the moderate N-cadherin dissociation is required for in vivo CCM through constraints.11 Therefore, it is needed to assess carefully how N-cadherin affects on the cancer cell migration in particular microenvironment such as three-dimensional (3D)extracellular matrix or in vivo. The non-small cell lung cancer PC14PE6 cell line was established previously22 and its value as an orthotopic model in nude mice was demonstrated.23,24 In a nude mice xenograft model, primary lesions are implanted by direct injection of cells into lung,25 which facilitates the identification of factors promoting lung cancer metastasis by using gene modification. In this study, we showed that alteration of LPP expression markedly changes the collectiveness and metastatic potential of cancer cells. LPP and its EMT partner, Etv5, inhibit N-cadherin-dependent adhesion by cooperatively activating matrix metalloproteinase 15 (MMP-15) expression, which directly cleaves N-cadherin extracellular domain. As a consequence of depletion of LPP, N-cadherin is upregulated, which promotes collective invasion and metastasis. We also found that there were some specimens having the heterogeneity of LPP expression, notably in some case, the expressions of LPP and N-cadherin were mutual exclusive in same primary lesion. Furthermore, we found that LPP expressions were missing in the distant metastatic lesions of adenocarcinoma specimens. Taken all together, loss-of-LPP in late step of cancer progression may

1 Department of Molecular Medicine and Biochemistry, Graduate School and Faculty of Medicine, Akita University, Akita, Japan; 2Department of Cellular and Organ pathology, Graduate School of and Faculty of Medicine, Akita University, Akita, Japan; 3Division of Medical Oncology, Cancer Therapeutics Development Program, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; 4Division of Genome Biology, Group for Development of Molecular Diagnostics and Individualized Therapy, National Cancer Research Center Institute, Chiba, Japan and 5Department of Thorasic Surgery, Akita University, Akita, Japan. Correspondence: Professor M Tanaka, Department of Molecular Medicine and Biochemistry, Akita University, Hondo 1-1-1, Akita, 010-8543, Japan. E-mail: [email protected] Received 25 July 2014; revised 13 March 2015; accepted 20 March 2015

Loss-of-LPP promotes collective migration S Kuriyama et al

2 trigger further dissemination and distant metastasis of lung adenocarcinoma. RESULTS Knockdown of LPP causes morphological changes via N-cadherin stabilization in lung cancer cell line, PC14PE6 and melanoma, Axcel First, to examine if LPP knockdown shows any morphological changes, we applied the LPP micro RNA (miR) expressing

retrovirus to various cell types (data not shown). The cell aggregation was observed in the non-small cell lung cancer cell line, PC14PE6 transfected LPP-miR vector (hereafter, LPP-miR cells) (Figure 1a, right), whereas the negative control miR-infected cells (Neg-miR cells) showed a round and non-adhesive morphology similar to that of the parent cell lines (Figure 1a, left). LPP-miR cells expressed the full-length N-cadherin, whereas Neg-miR cells have several cleaved forms of N-cadherin (Figure 1b). LPP is known to localize in FAs with similar LIM domain proteins such as zyxin or TRIP6,8,10 thus, we examined miR specificity in FA. LPP-miR

Figure 1. LPP knockdown alters the morphology and protein expression of N-cadherin in the non-small cell lung cancer (NSCLC), PC14PE6. (a) PC14PE6+negative control miR (Neg-miR) cell morphology (left) and LPP 8 miR-infected cells (right). (b) IBs of total cell lysates from Neg-miR and LPP 8 miR transfected cells. The cleaved band of N-cadherin was observed at 65 kDa. (c) LPP or vinculin localization with F-actin staining in PC14PE6. (d) IF (green) of ß-catenin (top), N-, (middle) and E-cadherin (bottom) in PC14PE6+ miR cells. Phalloidin staining (F-actin, magenta) shows cell shapes in merged images. (e) The diagram describes the positions of amplified N-cadherin complementary DNA (above). Weaker but full-size complementary DNA was amplified from Neg-miR sample (below). (f) Adding back myc-tagged LPP containing eight mutations of LPP 8 miR target sequences to LPP-miR cells. The restoring 66% of endogeneous LPP inhibits full-length N-cadherin. IB, immunoblot; IF, immunofluorescence; pAb, polyclonal antibody; mAb, monoclonal antibody. Oncogene (2015) 1 – 13

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Loss-of-LPP promotes collective migration S Kuriyama et al

3 specifically depleted LPP from FA, whereas vinculin expression in FAs were intact (Figure 1c). Next, Neg-miR and LPP-miR cells were cultured on the fibronectin in enough high cell density to make cell–cell adhesions. Neg-miR cells were weakly associated, but stained negative for ß-catenin and N-cadherin in cell–cell junctions, whereas LPP-miR cells showed strong positive staining for these proteins (Figure 1d). We also performed same experiment in melanoma cells using two independent miRs, and Axcel cell line, which does not have N-cadherin in normal culture condition, showed similar upregulation of N-cadherin by LPP-miRs (Supplementary Figures S1a and d). To see the generality of LPP role, we analyzed LPP expression in various lung adenocarcinoma cell lines (Supplementary Figure S1e). Despite LPP is ubiquitously found in lung adenocarcinomas, H1703 depleted LPP expression showed similar N-cadherin accumulation at the cell–cell junction (Supplementary Figures S1f and g). Next, to determine whether loss of full-length and presence of small fragments of N-cadherin in PC14PE6 cell was due to the alternative splicing, N-cadherin mRNA was analyzed by reverse transcriptase (RT)–PCR, which showed no splicing variant (Figure 1e). The overexpressed mycLPP containing eight mismatches to miR sequences canceled LPPmiR phenotypes (Figure 1f). Next, we checked if N-cadherin decrease is essential for morphological change of PC14PE6, then, N-cadherin knockdown in LPP-miR cells attenuated their clustering (Supplementary Figures S2a and c, S2e and g, S2i, S2k). Taken together, loss-of-LPP induces cell aggregation, which depends on the increase of N-cadherin. LPP inhibits N-cadherin through the regulation of nuclear localization of Etv5 LPP works as a co-transcriptional factor of Etv transcription factors.5,6 Etv1, Etv4 can bind to LPP6 and Etv5 is functionally associated.9 Although several Etv 1, 4 and 5 miR lentiviruses were tested to see the similar phenotype to LPP-miR, only Etv5-miR induced N-cadherin accumulation at cell–cell junctions and stabilized N-cadherin protein in western blot (Figure 2a, Supplementary Figure S2j). Moreover, endogenous Etv5 was distributed in nuclei of Neg-miR cells but not in LPP-miR cells (Figure 2a). The result was confirmed by nuclear translocation of transiently expressed EGFP (enhanced green fluorescent protein)tagged Etv5 protein in control PC14PE6 but it remains in cytoplasmic domain of the LPP-miR cell (Supplementary Figure S2l). These data indicate that LPP is required for Etv5 nuclei localization in PC14PE6, and LPP and Etv5 may act cooperatively to inhibit N-cadherin. Membrane-type metalloproteinase, MMP-15 is a common target of LPP and Etv5 Both LPP and Etv5-miR increased N-cadherin, however, it was not due to the alternative splicing of N-cadherin (Figure 1e). Therefore, full-length N-cadherin upregulation in LPP or Etv5-miR cells may be due to the inhibition of N-cadherin post-translational proteolysis. First, we tried to find common target from membranetype proteinases. ADAM10(ref. 26) and MMP-14 (MT1-MMP) are known to be involved in N-cadherin cleavage.27 It has also been known that MMP-14 is one of target of ERM (Etv5).28 However, ADAM10 was not affected, and MMP-14 was upregulated by LPP or Etv5 knockdown (Figure 2b, Supplementary Figure S2m). MMP-15 (MT2-MMP), which was first identified in the lung,29,30 was downregulated by both miRs (Figure 2b). We confirmed the decrease of MMP-15 by quantitative PCR and western blot (Figures 2b and c), also active MMP-15 in LPP and Etv5-miR cells (Figure 2c). In addition, MMP-15 was reduced in H1703+LPP-miR cells (Supplementary Figure S1e). To see if MMP-15 expression is essential for the morphological changes and N-cadherin expression at adhesion sites, gain- and loss-of-MMP-15 function were performed. The overexpression of mature MMP-15 (Δpro MMP-15) © 2015 Macmillan Publishers Limited

reduced N-cadherin-dependent cell adhesions in LPP-miR cells (Figure 2d, Supplementary Figures S2d and h), and two independent MMP-15 miRs reduced endogenous MMP-15, and increased N-cadherin (Figure 2e). These data suggest that MMP-15 is a common target of LPP and Etv5, and responsible for N-cadherin cleavage in PC14PE6. N-cadherin shedding is controlled directly by MMP-15 To test if MMP-15 cleaves N-cadherin, we performed in vitro digestion assays of the human N-cadherin extracellular domain and a mouse Fc protein fusion construct (hN-cadFc) with the purified MMP-15 proteins. The hN-cadFc was expressed in the U87MG glioblastoma cell line using adenoviral infection as described31 (Figure 2f). The HaloTag MMP-15 was produced in COS cells and purified by HaloTag-ligand-conjugated magnetic beads (Figure 2g), which could digest fibronectin as previously described32 (data not shown). Then, the hN-cadFc were incubated with or without MMP-15 bound magnetic beads for 0 or 20 h at 37 °C (Figures 2h and i). The naive hN-cadFc was observed at approximately 120 kDa, and the cleaved extracellular domain of N-cadherin, which was detected by an anti-rat N-cadherin antibody, showed a molecular weight of approximately 50 kDa (Figure 2h, arrowhead). The proximal portion of the Fc protein was detected at approximately 70 kDa in the presence of MMP-15 (Figure 2i, arrowhead), whereas MMP-15 alone did not show any bands (Figures 2h and i, lane 5). We confirmed the cleavage of N-cadherin Fc protein by cleavage of the purified commercial products for shorter incubation (Figure 2j). These results indicated that MMP-15 directly digests the N-cadherin extracellular domain. MMP-15 is directly regulated by the Etv5 transcription factor associated with LPP To confirm if LPP and Etv5 regulates MMP-15 expression, we analyzed gene regulatory regions of MMP-15 (Figure 3). A previous study showed that Etv recognizes AGAAAA sequences.33 We identified two Etv-binding sites in the upstream 2 kb of MMP-15 allele adjacent to GGAA sequences known as the Ets-binding site (Figure 3a). Compared with the minimal promoter, the -2 kb MMP-15 regulatory region (whole promoter) showed an approximately 10-fold increase of luciferase activity in Neg-miR cells (Figures 3b and c). Next, large deletions of Etv-binding domains (ΔR1, ΔR2) are tested (Figures 3a and b). ΔR2 showed decreased ratio in half of the whole promoter, whereas ΔR1 or the other’s change was moderate. These data suggest that the Etv binds mainly to element 2. To understand how LPP contributes MMP-15 regulation, we performed the same assay in LPP-miR cells. The increase ratio of the whole reporter in LPP-miR cells was similar to that of the ΔR2 reporter in Neg-miR cells (Figure 3c). These data suggested that LPP regulates MMP-15 expression through the mechanism involving Etv transcription factors. To further confirm whether element 1 or element 2 is involved in the regulation of MMP-15 expression, 6-bp of Etv-binding sites were deleted from full-length reporter. However, neither elements 1 nor 2 alone showed a significant reduction in activity. We then examined the effect of deletions of both elements 1 and 2, then, found that the level of Δele1, ele2 reporter activities dropped to that of ΔR2 mutant. These data suggest that Etv5 promotes MMP-15 likely with LPP as a co-factor, however, the sequences except Etvbinding site within R2 may be important for LPP function. Therefore, we next confirmed the binding of Etv and LPP to the MMP-15 regulatory region by chromosome immunoprecipitation (ChIP) assay (Figure 3d). We used LPP-miR cells expressing mycLPP, which restores MMP-15 expression (Figure 1f), to pull-down the chromosome DNA fragments by anti-Myc-tag (column 2), antiEtv5 antibodies (column 3) and non-related IgG as negative control (column 1) (Figure 3d), and performed quantitative PCR (qPCR) of each element (Figure 3a, arrows). From the amplification Oncogene (2015) 1 – 13

Loss-of-LPP promotes collective migration S Kuriyama et al

4 of ChIP-qPCR, we estimated the quantities of the pulled down chromatin fragments, and displayed as the fold increase (Figure 3d). To avoid cross-reaction of endogenous c-Myc, we

used human nucleostemin target sequence34 as negative control, and showed there is no amplification (Figure 3d, left). The graph showed that Etv5 dominantly binds to the element 2 rather than

Figure 2. LPP and Etv5 control MMP-15 and N-cadherin shedding. (a) IF of Neg, LPP 8 and Etv5#1 miR cells. N-cadherin (green) accumulations are seen in LPP 8 and Etv5-miR#1-infected cells (middle, right). Etv5 (red) nuclear localization is only observed in Neg-miR cells (left). (b) Semiquantitative RT–PCR analyses of membrane-linked metalloproteinases (above). The results of RT–qPCR of MMP-15 and ADAM-10 (graph). (c) IB of PC14PE6 cell lysates. Active-/MMP-15 protein levels were decreased in LPP or Etv5-miR cells. (d) Overexpression of HaloΔpro-MMP-15 in Neg/LPP/Etv5-miR cells. MMP-15 reduces full-length N-cadherin level and shows the cleaved bands. (e) Two independent miR for MMP-15 decreased MMP-15 and increase N-cadherin. (f) Purified hN-cadFc fusion proteins from adenovirus-infected U87MG cells. (g) Purified MMP-15 proteins from COS-1. (h) In vitro cleavage of hN-cadFc with MMP-15 beads. IB with N-cadherin extracellular domain antibody. Rat secondary antibody does not cross-react with mouse IgG. N-terminal side of the cleaved band was approximately 50 kDa. (i) The same samples as (h). IB with mouse IgG. C-terminal side of the cleaved band was approximately 70 kDa. (j) The commercial N-cadFc (R&D) digestion for shorter ( o6 h) incubation times. Immunoblot with N-cadherin extracellular domain antibody reveals that 1- h incubation showed cleaved proteins. bds, beads; CS, crude samples; EL, eluates; N-cadh, N-cadherin; α-Tub, alpha-tubulin. Oncogene (2015) 1 – 13

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Figure 3. Etv5 and LPP directly bind to MMP-15 upstream and regulate MMP-15 expression cooperatively. (a) Schematic diagram of the human MMP-15 gene structure around exon 1. The red box indicates the Etv-binding site, AGAAAA. The blue box indicates the Ets-binding site, GGAA. (b) Reporter construct and its mutants. (c) Average fold increase obtained from dual-luciferase assays using the firefly reporter shown in b and Renilla luciferase internal controls. The values are normalized to minimal promoter-luciferase activities; one-way analysis of variance (ANOVA) P o0.0001, individual comparison **P o0.05 among Neg-miR samples, one-way ANOVA P = 0.0056, individual comparison *P o0.05 among LPP-miR samples. (d) ChIP-quantitative PCR (qPCR) analysis of the Etv-binding sites (elements 1 and 2) in the PC14PE6+LPPmiR+myc-LPP cell line. The graph shows the relative values of the estimated amount of template DNAs pulled down by non-related-Ab (left, negative control of pull-down), Myc-Ab (LPP, middle) and Etv5-Ab (right, Etv5). Human c-Myc target gene, nucleostemin (hspNS) primer was used for negative control of qPCR. (e) Etv5 binds to element 2. LPP associates with Etv5 and the unidentified sequences within region 2. ANOVA, analysis of variance.

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7 element 1, and LPP may not be directly bound with Etv-binding site, but likely associated to the adjacent sequences within the region 2 (Figure 3e). The strong binding of LPP to element 2 was confirmed by ChIP using LPP antibody (Supplementary Figure S3). These data suggested that the Etv5-LPP complex binds directly to the MMP-15 gene regulatory region in PC14PE6 cells. Depletion of LPP-Etv5 increases their invasiveness in a collagen gel containing cancer-associated fibroblasts Increased N-cadherin in LPP-miR cells promoted collectiveness (Figure 1); however, their cell migration ability and invasiveness as a collective cellular sheet have not been analyzed. Therefore, we investigated how LPP/Etv5-miR cells behave, then performed an in vitro 3D collagen gel invasion assay using cancer-associated fibroblasts (CaFs)35 (Figure 4). LPP or Etv5-miR cells showed collective invasion into collagen gels (Figures 4b and c), whereas Neg-miR cells did not show the large invasive protrusions (Figure 4a). As the metastasis of lung cancer is often promoted by epidermal growth factor (EGF),36 we added EGF into the medium in the lower chamber to analyze their invasiveness. LPPmiR cells and Etv5-miR cells invaded more to the gel containing CAF with EGF, compared with only CAF (Figures 4b’ and c’), whereas Neg-miR cells did not increase their invasiveness by EGF (Figure 4a’). Quantitative analysis of the area of invaded clusters under each condition (Figure 4d) confirmed that LPP or Etv5 knockdown increased cell invasiveness. Adding back LPP to PC14PE6+LPP-miR cells reduced the invasiveness (Supplementary Figure S4c). Furthermore, the sections of collagen gels are also stained by N-cadherin immunohistochemistry (IHC) using extracellular domain-specific antibody (Figures 4e and g’). The invaded groups and cellular sheet of LPP or Etv5-miR cells express extracellular N-cadherin at cell–cell junctions, despite Neg-miR cellular sheet showed only limited stains (Figures 4e and g’, arrowheads). Furthermore, N-cadherin knockdown in LPP-miR or Etv5-miR cells revealed that N-cadherin is essential for the collective invasion (Supplementary Figures S4d and f, S4k and p). The overexpression of MMP-15 decreases the invasiveness of LPP-miR cells (Supplementary Figure S4g). The second Etv5-miR also increases the invasiveness (Supplementary Figures S4h and i). These data indicate that restoring whole N-cadherin protein in lung cancer cells promotes invasive phenotypes. More generally, in Axcel melanoma (Supplementary Figures S1h and i) and in the other lung adenocarcinoma H1703 (Supplementary Figures S4q and r), N-cadherin accumulating cells by LPP-miR increased their migrations. Loss-of-LPP promotes metastasis of PC14PE6 cells by orthotopic implantation model To see if LPP depletion promotes invasion and dissemination in vivo, we investigated the spreading of the tumor by orthotopic implantation of PC14PE6 cells as cell clumps in mice lung (Figure 5). We made the traceable PC14PE6 cell lines with mCherry and luciferase by in vivo imaging, and these cells formed detectable lesions in the injected lungs 1 week after injection. The luminescent signals of metastasized LPP-miR cells in the

opposite lung were detected after 14 to 21 days (Figure 5b), whereas Neg-miR cells remained in the injected lung (Figure 5a, Table 1). The tumor nodules in lungs were confirmed in the dissected lung of Neg or LPP-miR-cells injected mice (Figures 5c and d, arrowheads). We also analyzed the opposite lungs of the injected lungs using mCherry fluorescence, not only large nodules but also many LPP-miR-cell clusters in the opposite lung were observed, whereas no red Neg-miR cell was observed (Figures 5e and f, arrows), indicating that LPP-miR cells were diffusely disseminated in the opposite lung within 3 weeks (Table 1). To see if N-cadherin accumulation can be detected in vivo, we performed N-cadherin IHC (Figures 5g and h). An intense N-cadherin signal was observed in the LPP-miR cell tumor (Figure 5h), whereas N-cadherin was not seen in Neg-miR cell tumor (Figure 5g). We confirmed that the disseminations of the cells to the opposite lung were blocked by adding back LPP to LPP-miR cells (Supplementary Figures S5a and f). Knockdown of N-cadherin reduced survival of tumor in the primary lesion and blocked the dissemination of cells into opposite lung, and the forced expression of MMP-15 reduces large nodules and overall percentage of cancer dissemination (Supplementary Figures S5g and j, Table 2). We also tested mRNA elevation of N-cadherin upon the dissemination. The elevated synthesis of N-cadherin was not occurred in disseminated lesions (Supplementary Figures S5k and l). These data indicate that the stabilization of N-cadherin cell adhesion complexes in lung cancer accounts for the different degrees of metastasis. LPP expression in various lung cancer specimens The expression of LPP in lung cancer was investigated by IHC of tissue array of surgically obtained specimens (Figures 6a–c, Table 3). LPP is strongly expressed in smooth muscle,37 thus the IHC signals appeared in the region of normal tissues. According to smooth muscle staining, we could assess signal intensities of LPPIHC. Intense staining of LPP was observed in adenocarcinoma and in squamous cell cancer (Table 3, Figure 6b). LPP was not detected in small cell lung cancer specimens (Table 3, Figure 6c). Only few specimens of the other types of lung cancer such as pleomorphic adenoma, large cell neuroendocrine carcinoma were stained (Table 3). LPP heterogeneity coincides with N-cadherin accumulation in lung adenocarcinoma As LPP was detected at high incidence in lung adenocarcinoma, and MMP-15 is also known to be overexpressed in lung adenocarcinoma,30 we further examined precise expression of LPP in surgical specimens of lung adenocarcinoma. In some cases, we found heterogeneity of LPP expressions within the primary tumors, which was complementary to N-cadherin expression (Figures 6d–f). This mutual exclusiveness of N-cadherin against LPP-rich region were found in 4 specimens of 16 specimens having LPP heterogeneity, which were found from 27 surgery specimens we tested. The detail information of these specimens are listed in Supplementary Table S1. The pathological marker of lung adenocarcinoma, TTF-1 and MIB-1 were tested on these

Figure 4. PC14PE6 Neg, LPP and Etv5-miR cells on CAFs (CaF-37) mixed collagen gels showed their invasiveness. (a–c) Cell invasion toward CaF-containing gel. (a’–c’) Cell invasion toward CaF-containing gel enhanced by additional EGF (10 ng/ml) in the medium of lower chamber. (a and a’) Neg-miR cell invasion. (b and b’) LPP-miR cells invaded into the gel as a large cohort. (c, c’) Etv5-miR cells also showed invasion into the gel. (d) Quantitative analyses of the region invaded by cells. The random and individual squares (n = 15) were chosen, which includes one cell diameter of the cellular sheet and the invaded cells. The color was adjusted as black and white image, and quantified cell areas by Image J (v1.44, National Institutes of Health, Bethesda, MD, USA). The non-invasive cellular sheet has the minimum area values, thus, we divide the values of the measured area of LPP or Etv5-miR. The graph indicates the fold increase of the invaded cells area. The error bars indicate s.e. (e–g) The IHC of Neg/LPP/Etv5-miR cells with or without EGF stimulation (e’–g’). (e and e’) Neg-miR cells. (f and f’) LPP-miR-8 cells. (g and g’) Etv5-miR#1 cells by MNCD2 extracellular N-cadherin antibody (HDB). Arrows indicate N-cadherin IHC signals in the cohorts. © 2015 Macmillan Publishers Limited

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Figure 5. In vivo orthotopic implanted LPP-miR cells disseminated to the opposite lung. (a and b) Heat map of IVIS images generated by detection of luciferin glowing PC14PE6 cells. Different time points in the same mouse are shown (above to below). Two different examples are indicated. P stands for primary, D stands for disseminated side of the experiments. (a) Neg-miR +luc2 IRES mCherry co-infected cells were injected into the lungs of mice. (b) LPP-miR, luc2/mCherry were injected into the right lung and the luminescence was detected in the left side 21 days after the injection. (c and d) Dissected lungs of primary (lower) and disseminated lesions (upper). (e and f) The mCherry labeled cells indicate that the injected cells migrated into the opposite lung of the injected lung. Arrows indicate small clusters of cells. (e) No dissemination is found in the opposite lung of the Neg-miR cell injected lung. (f) Multiple nodules (N) and small patches (arrows) are observed. (g and h) N-cadherin IHC of the primary lesions. (g) Neg-miR tumors or the surrounding connective tissues were faintly stained. (h) The rosettes of N-cadherin staining were observed in LPP-miR tumors.

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Table 1. The results of IVIS imaging of the orthotopic injection of PC14PE6 into lung of male nude mice Marker-positive lung Neg-miR LPP-miR

Injected lung (+)/counter (− ) Both lungs (+) Injected lung (+)/counter ( − ) Both lungs (+)

o20 Days

35 Days*

9 0 4 11

0 1 NT NT

Abbreviations: LPP, lipoma preferred partner; miR, micro RNA; NT, not tested. Cancer cells were injected into left or right lung, and from day 6, luciferase activities were monitored by IVIS imaging system, then the leaking could be detected. Counting the number of mice that had luciferase signals only injected lung for a while, and later the cancer cells were disseminated. The mice had the leaking signals had been removed from the counts. The experiments were repeated three times. PC14PE6+Neg-miR cell injected mice (total n = 9) showed no dissemination for 20 days. PC14PE6+LPP-miR cell injected mice (total n = 15) showed strong dissemination (73.3%). *The mouse had luciferase signal in the injected lung at day 20 finally became to show the positive signal in the opposite lung at day 35.

Table 2.

The results of injections of the various rescue cell lines Number of nodules in oppsite lung

Neg miR LPP miR8 +myc LPP +Ncad miRs +MMP-15

n

Pri

Dis

%

Avg L size

Avg S size

7 7 9 11 12

7 7 8 5 12

0 7 1 0 7

0 100 13 0 58

0 1.57 0 0 0.08

0 3.71 0.11 0 2.92

Abbreviations: LPP, lipoma preferred partner; miR, micro RNA; MMP-15, matrix metalloproteinase 15. The number of mice we injected is shown in n. ‘Pri’ is the number shown tumor growth in the injected lungs. ‘Dis’ is the number shown any disseminated lesions in the opposite lungs. The numbers of the different sizes of nodules are counted and averaged. The L size nodules are 43 mm. S size nodules are from 0.5 to 3 mm diameter. The average L size nodule number of LPP miR8 samples is 1.57 (s.e. ±0.15), and S size is 3.71 (s.e. ± 0.31). The average S size of LPP miR8+mycLPPmut8 is 0.11 (s.e. ± 0.03). The average L size number of LPP-miR+MMP-15 samples is 0.08 (s.e. ± 0.02), and S size is 2.92 (s.e. ± 0.31). The differences of average L size nodules were significant (Po0.01).

specimens, and we found that N-cadherin-positive area is overlapped with TTF-1 or MIB-1 (Supplementary Figures S6a and h). The quantification of N-cadherin-positive cell ratio in LPP-rich or -poor cells within these specimens indicates mutual exclusiveness is significant in these particular specimens (Supplementary Figure S6i). To know whether the loss-of-LPP coincided with metastasis of adenocarcinomas, we examined IHC staining of three sets of primary and distant metastasis specimens from lung adenocarcinoma patients (Figures 6g–k, Supplementary Figures S6j and p). In three of three cases, the intensities of LPP-IHC in distant metastatic lesions were lower than that of primary lesions (Supplementary Figures S6j and p). Particularly, in some case, LPP was abolished in opposite lung metastasis and kidney metastasis (Figures 6g–i), which is compatible with our experimental results (Figures 4 and 5). Taken all together, loss-of-LPP is well correlated with the increase of the collectiveness of cancer cells, and subsequently leads distant metastasis, therefore, LPP would be a novel prognostic marker of distant metastasis. © 2015 Macmillan Publishers Limited

DISCUSSION CCM of cancer requires cadherin-based cell junction making special cellular properties such as collective polarity, force generation and cooperative functions.38 We demonstrated for the first time that N-cadherin accumulations by LPP depletion in the groups of cancer cells increase their invasion in 3D culture and dissemination in vivo xenograft. Furthermore, we preliminary demonstrated that the occasional loss-of-LPP increased N-cadherin expression, which give us a new interpretation of considering the heterogeneity of cancer cells. LPP and Etv5 cooperatively activate ZEB1 expression and promote EMT of endometrial cancer cells.6 Cancer EMT often triggers de novo expressions of N-cadherin and metalloproteinases, and cancer cells exhibits individual migration and cleavages of extracellular matrix to go out from the primary lesions.39,40 However, PC14PE6 cells usually repressed N-cadherin by LPP, which means that EMT process has not yet complete in these cells. Furthermore, the loss-of-LPP increases their invasiveness in vitro 3D culture, despite the whole level of MMPs were decreased in gelatin zymography (Supplementary Figures S7a and c) and major gelatinases are not precisely controlled by LPP (Supplementary Figure S7b). Then, how can LPP-depleted PC14PE6 cells invade into collagen gel without overexpression of extracellular matrix proteinases? In collagen-rich 3D gel invasion, CCM requires protein cleavage of extracellular matrix.38–41 Particularly, melanoma or fibrosarcoma cells alter their invasion mode dependent on 2.5 to 8 mg/ml collagen concentrations, and show collective behavior in relatively higher concentration of collagen gel in presence of MMPs.42 In the 3D invasion assay of MDCK cells, which have undergone EMT by hepatocyte growth factor, elevated N-cadherin expression enables the cells to migrate in 1 mg/ml collagen gel as multicellular chain.43 In our assay, we started 2.4 mg/ml of collagen to make gels, thus, the condition of our gel invasion assay is somewhere in the middle of these observations, thus, N-cadherin-dependent collectiveness in LPP-miR cells is likely the way to invade into collagen gel. LPP may be required for EMT-mediated early steps of cancer progression, but loss-of-LPP leads to dissemination and distant metastasis. N-cadherin would be a most important molecular property for CCM because it is necessary not only for migration itself,15,44 but also for the cell cooperation21 or contact inhibition.31 In addition to them, the precise regulation of N-cadherin under LPAR2 control is also required for CCM especially through the interstices of surrounding tissues.11 The LPP-depleted PC14PE6 cells, which abnormally restore full-length N-cadherin, did not jam into collagen gel in vitro or primary lesions in vivo, increase their ability to migrate into the dense structure. In future study, the comparison of phenotypes between LPAR2 and LPP, if any in the same system, will be interesting. We found that there is the heterogeneity of the level of LPP over each specimen of adenocarcinoma, and the mutually exclusive expressions of LPP and N-cadherin can be observed inside of the primary lesions (Figures 6d–f). The clonal evolution by DNA errors may produce the intratumor heterogeneity.45 The loss-of-LPP can be observed in the distant metastatic lesions (Figures 6g–k). So far, we do not know how LPP is turned off upon the metastasis. In smooth muscle cells, LPP is strongly expressed, and the expression is controlled by serum response factor.46 In future, it is interesting to see if serum response factor expression or upstream of serum response factor is altered in the specimens having LPP heterogeneity. We tried to see N-cadherin expression in the distant metastatic lesions, however, none of sample can be stained by N-cadherin IHC. It is also possible that the metastasized cancer cells start to express the other cadherin to undergo mesenchymal-to-epithelial transition,47 otherwise, N-cadherin is transiently expressed only during the migration. However, we preliminary found the possible Oncogene (2015) 1 – 13

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10 explanation of these observations. Valproic acid is known bipolar disorder medicine, later known as a histone deacetylase inhibitor.48,49 Valproic acid promotes hyper acetylation of the promoters even in cancer cells.49 PC14PE6 cells markedly alter

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their morphologies when valproic acid was added in culture medium (Supplementary Figures S7d and e). Notably, N-cadherin accumulation happened by valproic acid treatment means that N-cadherin expression is usually repressed by epigenetic

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11 modifications in PC14PE6 cells (Supplementary Figure S7f). According to large comprehensive research of lung adenocarcinoma, alternations of LPP/Etv5/MMP-15/N-cadherin pathway are found in 27.4% (Supplementary Figure S8, 63/230, TCGA, lung adenocarcinoma, Nature 2014).50 However, we found that the features of only two specimens fit to our phenotypes (Supplementary Figure S8, arrows). The reason why we could not see the mutual exclusiveness of N-cadherin vs LPP in TCGA database would be due to the heterogeneity of LPP expressions. Our results implies the future evaluations of this pathway’s upstream, epigenetic modifications and heterogeneity in multiple foci within the tumors may provide relevant prognosis of metastasis in lung adenocarcinomas. MATERIALS AND METHODS Cell lines

Immunofluorescence and confocal microscopy The staining procedure was described in previous paper.11 Antibody information was written in supplemental text. The images were obtained by LSM510META or LSM780 (Zeiss, Oberkochen, Germany) confocal microscope, and processed in Zen software (Zeiss) and Adobe Photoshop CS.

The signal intensities of LPP-IHC in cancer patient specimens Signal intensity 2+

Squamous cell carcinoma Adenocarcinoma Small cell lung cancer Pleomorphic LCNEC

The specimens of tissue array and the individual sections were obtained from the patients who underwent pneumonectomy or lobectomy without preoperative chemotherapy or radiotherapy. All samples were collected from the surgical pathology files at Akita University Hospital, Akita, Japan, between 2005 and 2011 and the tissues were obtained with the informed consent of the patients according to Akita University ethical committee for medical researches. The specimens for immunohistochemical evaluation were taken from representative areas of each tumor, including the largest cut surface.

Immunohistochemistry The staining and coloring procedures are described in elsewhere of the previous paper.35 For activation of antigen, we used high pH treatment (Dako, Glostrup, Denmark).

HaloTag purification and in vitro digestion

PC14PE6 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics. The melanoma cell line, Axcel, was established from metastatic lesions of melanoma patients by Dr Yuichiro Hamanaka (unpublished data), and Axcel and all lung adenocarcinomas were cultured in RPMI-1460 with 10% fetal bovine serum. The 37CaF35 was cultured in high glucose Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Modified cell lines with retrovirus constructs (see Supplementary Materials and methods) were cultured under the same conditions as the original cell lines with the selection antibiotics G418 sulfate (800 μg/ml, Wako, Osaka, Japan), puromycin (1 μg/ml), and hygromycin B (400 μg/ml, Wako).

Table 3.

Specimens from cancer patients

9/36 82/144 0/4 1/7 1/4

(25%) (57%) (0%) (14%) (25%)

Signal intensity 3+ 0/36 19/144 0/4 0/7 0/4

(0%) (13%) (0%) (0%) (0%)

Abbreviations: IHC, immunohistochemistry; LCNEC, large cell neuroendocrine carcinoma; LPP, lipoma preferred partner; SM, smooth muscle. Cancer patient specimens of primary lesions are sectioned and stained with LPP-IHC. The signals were developed with DAB chromogen. The degrees of signal intensities are judged from the staining within the SM tissues, which are always quickly developed and most strongly stained. Immunoreactivity was scored semiquantitatively according to the intensity (1+, detectable, but faint; 2+, evident, but weaker than the intensity in smooth muscle of blood vessels; 3+, equal to the intensity in the smooth muscle of blood vessels).

The human MMP-15 complementary DNA lacking pro-domain region was directly cloned by RT–PCR of PC14PE6 cells’ RNA, and inserted to signal peptide-HaloTag vector previously made.11 The COS lysates of HaloTagΔpro MMP-15 protein was purified by Mammalian Pulldown system (Promega, Madison, WI, USA). After washing three time, the MMP-15 beads were briefly rinsed with in vitro digestion buffer, added the substrates and incubated for 0–20 h.

Luciferase assay The human MMP-15 gene regulatory region (−2 kb MMP-15) was amplified by genomic PCR and subcloned into the luciferase vector (−2 kb MMP-15min P-luc2), and Etv-binding sites were modified by PCR (see Figures 3a and b). The 1.8 μg of luciferase vector and 0.2 μg of pGL4.74 (hRluc) were transferred into 5 × 105 cells of PC14PE6 using the Ingenio Electroporation Kit (Mirus Bio LLC, Madison, WI, USA). The conditions for electroporation were 135 V, 50 ms, 2 pulses (0.2 cm cuvette, Nepa21edit, Nepagene, Chiba, Japan). After 2 days of transfection, cells were analyzed using the DualLuciferase Assay Kit (Promega) and GeneLight luminometer (GL-200, Microtec Co., Ltd, Chiba, Japan). The raw values of relative light unit (firefly)/relative light unit (Renilla) were normalized by minimal promotor’s values. We performed one-way analysis of variance, and t-test for particular sets of results.

Collagen gel invasion assay The collagen gel invasion assay was performed as previously described.51 The 37CaF was used as the provider of attractants. Dulbecco’s modified Eagle’s medium+10% fetal bovine serum with/without 10 ng/ml EGF was fulfilled in the cell strainers (lower chamber), the disks were cultured for 7 to 14 days, and embedded in paraffin, then sectioned.

Xenograft model and in vivo imaging To monitor tumor growth and metastasis, mCherry-IRES2-luciferaseinfected cells were established for each cell line, PC14PE6 cells were injected into the lungs of male nude mice (Balb/c, nu/nu, 5-week-old) using 29G needle syringes containing 1 × 106 cells/20 μl Matrigel mixture. The tumors were monitored by aLuciferin (250 μl/25 g nude mouse, 15 mg/ml in phosphate-buffered saline) (Avidin Ltd, Szeged, Hungary) and the IVIS system (Perkin-Elmer, Waltham, MA, USA) for 3–4 weeks. The animal experiments are approved by Akita University’s ethical committee for the experimental animals.

Figure 6. The IHC of surgical specimens of lung cancer patients probed against anti-LPP antibodies. (a–c) Various lung cancer tissue array samples. (a) Adenocarcinomas were strongly positive (3+). (b) Squamous cell carcinomas were moderately positive (2+). (c) Small cell lung cancer specimens were negative. (a’–c’) Higher magnifications (x40). (d–f) Heterogeneity of N-cadherin or LPP expressions in lung adenocarcinoma patient’s specimen. (d) Hematoxylin-eosin (HE) image of primary tumors similar to 6e and f. (e) LPP-IHC. (f) N-cadherin IHC. N-cadherin and LPP expressions are mutually exclusive in this area. (g–i, j and k) The sets of primary and metastatic lesions of adenocarcinoma patient specimens stained with LPP-IHC. (g) Primary lesion of LPP-IHC in high magnification (x100). (h) The cancer cells (CCs) in opposite lung metastasis of same sample of g. To avoid confusion of signals in fibroblast cells, cells are observed in high magnification. (i) Kidney metastasis of LPP-IHC. (j) Primary lesion of another specimens. (k) Brain metastasis. Small vein (VN) integrated into metastatic lesion is stained positive. Surrounding CCs were negative. © 2015 Macmillan Publishers Limited

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12 ChIP-quantitative PCR and RT–qPCR The ChIP assay was partially modified from the procedure described.52 DNAs were pulled down by Myc-tag antibody (9E10X, 1:60, Santa Cruz, Dallas, TX, USA), anti-Etv5 antibody (Cell Applications, San Diego, CA, USA, CP10288, 1:120) and anti-hPleckhn1 antibody (T-13, 1:50, Santa Cruz) or anti-EGFP (Abnova, Taipei, Taiwan) as non-related IgG. As a negative control for qPCR, the human Myc target gene, nucleostemin primer set was used as non-related genomic primers, as previously described.34 The precipitated DNAs (ChIP-qPCR) or complementary DNAs (RT–qPCR) were amplified using DNA Fast Green Master mix (Roche, Mannheim, Germany), and analyzed by Lightcycler Nano (Roche). The error bars were automatically produced by Lightcycler Nano software (Roche).

Supplemental materials and methods Gene knockdown reagents, virus particles, RT–PCR procedures, primer sequences, antibody information and buffer compositions are described in supplemental text.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Dr Yuichiro Hamanaka for kindly providing the melanoma cell lines. The MNCD2 antibody developed by Takeichi and Matsunami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by Department of Biology, The University of Iowa, Iowa City, IA, USA. This work is supported by JST KAKENHI (grant number 24700962, SK; grant number 25290042, 26640068, MT) and MEXT KAKENHI (grant number 25111702, SK).

AUTHOR CONTRIBUTIONS SK performed the experiments. SY and TK provided the set of lung cancer cell lines. MY, YM and AG made human cancer patient specimens. NA, MT and AG analyzed IHC of patient specimens. SK and MT designed the experiments, and wrote the manuscript.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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