Gremlin-1 associates with fibrillin microfibrils in vivo and regulates mesothelioma cell survival through transcription factor slug

Abstract

Malignant mesothelioma is a form of cancer that is highly resistant to conventional cancer therapy for which no major therapeutic advances have been introduced. Here, we identify gremlin-1, a known bone morphogenetic protein inhibitor crucial for embryonic development, as a potential therapeutic target for mesothelioma. We found high expression levels of gremlin-1 in the mesothelioma tumor tissue, as well as in primary mesothelioma cells cultured from pleural effusion samples. Downregulation of gremlin-1 expression by siRNA-mediated silencing in a mesothelioma cell line inhibited cell proliferation. This was associated with downregulation of the transcription factor slug as well as mesenchymal proteins linked to cancer epithelial-to-mesenchymal transition. Further, resistance to paclitaxel-induced cell death was associated with high gremlin-1 and slug expression. Treatment of gremlin-1-silenced mesothelioma cells with paclitaxel or pemetrexed resulted in efficient loss of cell survival. Finally, our data suggest that concomitant upregulation of fibrillin-2 in mesothelioma provides a mechanism for extracellular localization of gremlin-1 to the tumor microenvironment. This was supported by the demonstration of interactions between gremlin-1, and fibrillin-1 and -2 peptides as well as by colocalization of gremlin-1 to fibrillin microfibrils in cells and tumor tissue samples. Our data suggest that gremlin-1 is also a potential target for overcoming drug resistance in mesothelioma.

OPEN

Citation: Oncogenesis (2013) 2, e66; doi:10.1038/oncsis.2013.29& 2013 Macmillan Publishers Limited All rights reserved 2157-9024/13

http://www.nature.com/oncsis

Web End =www.nature.com/oncsis

ORIGINAL ARTICLE

Gremlin-1 associates with brillin microbrils in vivo and regulates mesothelioma cell survival through transcription factor slug

JA Tamminen1,2, V Parviainen2, M Rnty3,4, AP Wohl5, L Murray1,2, S Joenvaara2, M Varjosalo6, O Lepparanta7, O Ritvos3,8, G Sengle5, R Renkonen2,3, M Myllarniemi7 and K Koli1,2

Malignant mesothelioma is a form of cancer that is highly resistant to conventional cancer therapy for which no major therapeutic advances have been introduced. Here, we identify gremlin-1, a known bone morphogenetic protein inhibitor crucial for embryonic development, as a potential therapeutic target for mesothelioma. We found high expression levels of gremlin-1 in the mesothelioma tumor tissue, as well as in primary mesothelioma cells cultured from pleural effusion samples. Downregulation of gremlin-1 expression by siRNA-mediated silencing in a mesothelioma cell line inhibited cell proliferation. This was associated with downregulation of the transcription factor slug as well as mesenchymal proteins linked to cancer epithelial-to-mesenchymal transition. Further, resistance to paclitaxel-induced cell death was associated with high gremlin-1 and slug expression. Treatment of gremlin-1-silenced mesothelioma cells with paclitaxel or pemetrexed resulted in efcient loss of cell survival. Finally, our data suggest that concomitant upregulation of brillin-2 in mesothelioma provides a mechanism for extracellular localization of gremlin-1 to the tumor microenvironment. This was supported by the demonstration of interactions between gremlin-1, and brillin-1 and -2 peptides as well as by colocalization of gremlin-1 to brillin microbrils in cells and tumor tissue samples. Our data suggest that gremlin-1 is also a potential target for overcoming drug resistance in mesothelioma.

Oncogenesis (2013) 2, e66; doi:http://dx.doi.org/10.1038/oncsis.2013.29

Web End =10.1038/oncsis.2013.29 ; published online 26 August 2013

Subject Categories: Molecular oncology

Keywords: gremlin; brillin; mesothelioma; EMT; slug

INTRODUCTIONMalignant mesothelioma is an aggressive tumor, which originates from the mesothelial surface cells lining the serous body cavities such as the pleura, peritoneum or pericardium.1 Mesothelioma is strongly linked to asbestos exposure, and it may take several decades to develop after initial exposure.2 Because of the long latency, the incidence of mesothelioma will increase in the near future worldwide.3,4 Mesothelioma is resistant to chemo- and radiotherapy, leading to poor prognosis for patients suffering from this malignancy.4 New markers for screening and monitoring the disease and in particular new drug targets are needed.

Cancer progression is associated with a re-expression of developmental programs, which contribute to the proliferative and invasive properties of tumor cells.5,6 Gremlin-1 is a cysteine knot protein, which belongs to the DAN family of bone morphogenetic protein (BMP) antagonists. Gremlin-1 can bind to and inhibit the functions of BMP-2, -4 and -7.7,8 These three BMP isoforms are targeted to brillin microbrils, suggesting extracellular regulation of bioavailability.9 Gremlin-1-mediated BMP antagonism is crucial for mouse lung and kidney development.10 In adult mouse and human tissues gremlin-1 expression is low. Originally, gremlin-1 was identied as a gene downregulated in v-mos-transformed broblasts and therefore

called Drm.11 However, recent studies suggest the overexpression of gremlin-1 in epithelial cancers including lung carcinomas.12,13

The role of gremlin-1 in cancer has remained largely unclear. As our preliminary results suggested an upregulation of gremlin in mesothelioma tissue, we set out to nd molecular interactions of gremlin and study the biological function of gremlin in mesothelioma.

RESULTSGremlin-1 interacts with brillin-1 and -2

A search for new gremlin interacting proteins was carried out using systematic afnity purication coupled with mass spectrometry identication (AP-MS). We used the well-characterized Flip-In-HEK293 cell system to produce C-terminally tagged gremlin-1 and a standardized workow for the purication of protein complexes from cell lysates14 (Figure 1a). A low tetracycline concentration (25 ng/ml) was selected for gremlin-1 induction (Figure 1b). Immunoblotting analyses after different afnity purication steps suggested that gremlin was well recovered (Figure 1c).

In order to characterize the interactome of gremlin-1, immunopuried proteins were identied using mass spectrometry. Proteins

1Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland; 2Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland; 3Helsinki University Central Hospital and Hospital Laboratory, Helsinki, Finland; 4Department of Pathology, University of Helsinki, Helsinki, Finland; 5Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany; 6Institute of Biotechnology, University of Helsinki, Helsinki, Finland; 7Division of Pulmonary Medicine, Department of Medicine, University of Helsinki, Helsinki, Finland and 8Department of Bacteriology and Immunology, University of Helsinki, Helsinki, Finland. Correspondence: Dr K Koli, University of Helsinki, Biomedicum/B502a1, PO Box 63, Haartmaninkatu 8, Helsinki 00014, Finland.

E-mail: mailto:[email protected]

Web End =katri.koli@helsinki. Received 10 July 2013; accepted 16 July 2013

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Figure 1. Gremlin-1 interacts with brillin-1 and -2. (a) Gremlin-1 protein interaction screen outline. (b) Flip-In HEK293/gremlin-1 cells were treated with increasing concentrations of tetracycline for 24 h followed by immunoblotting analyses using anti-HA antibodies. Ponceau staining of proteins indicates equal loading. (c) Immunoblotting analyses of gremlin-1, using anti-HA antibodies, after Strep-tactin and anti-HA afnity purication steps. (d) Purication of gremlin-1 from conditioned media of stably transfected HEK293 cells. After elution from a CoCl2-loaded HiTrap column using a C-terminally placed His6-afnity-tag protein, bands corresponding to monomeric, dimeric and multimeric gremlin-1 were detected via SDSPAGE followed by Coomassie staining. A subsequent second purication step via a HiTrap heparin column yielded mostly dimeric gremlin-1, which shifted to the position of monomeric gremlin under reducing conditions ( SH). (e) Interaction

studies between gremlin-1 and N-terminal peptides of brillin-1 and -2 using a surface plasmon resonance technology. Gremlin-1 was immobilized on a sensor chip and brillin-1 and -2 peptides in a concentration range from 80 to 0 nM were own over as analytes. Afnity constants (Kd) for both interactions were about 10 nM (Table 1), indicating a high afnity interaction between gremlin-1 and the main building blocks of brillin microbrils.

Table 1. Surface plasmon resonance afnity data for tested interactions between gremlin-1 and brillin-1 and -2 N-terminal peptides

Interaction (ligand/analyte) kon (1/M*s)/koff (1/s) Kd (nM)

gremlin-1 dimer/ brillin-2 (rF86)

2.41 105/

2.18 10 3

were rst digested using trypsin and then fractionated with reverse-phase chromatography online using a mass spectrometer. The resulting peptide spectra were identied with two different search engines. A total of 12 proteins were identied to interact with gremlin-1 in three replicate experiments and with both search engines. The interaction data were ltered using a protein list (data not shown) of 377 unspecic binding proteins acquired using Flip-In-HEK293 cell line transfected with the afnity tag containing plasmid. The nal ltered list contained four proteins: cytokeratin-9 (Swiss-Prot: P35527), brillin-2 (Swiss-Prot: P35556), cytokeratin-2a (Swiss-Prot: P35908) and APOBEC1-binding protein 2 (Swiss-Prot: Q9UBS4). Fibrillin-2 was identied in all three replicate experiments and with both search engines, which improves the condence of the identication. In addition, brillin-2 was identied with up to three distinct and good scoring peptides in two of the replicates further validating the interaction of brillin-2 and gremlin-1. Fibrillins are constituents of extracellular microbrils and have a role in elastin assembly.15 They also sequester and regulate the bioavailability of growth factors such as BMP isoforms.9

In order to conrm our ndings, we tested direct interactions between puried gremlin-1 and recombinant N-terminal peptides of brillin-1 and -2 in proteinprotein interaction assays using surface plasmon resonance technology. When puried gremlin-1 (Figure 1d) was used as a ligand immobilized on the sensor chip surface and N-terminal brillin were own over as analytes direct interactions with molecular afnities in the low nanomolar range (Table 1) could be measured (Figure 1e). This suggests a high

9.05

gremlin-1 dimer/ brillin-1 (rF11)

1.92 105/

1.45 10 3

7.55

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molecular interaction between gremlin-1 and the two main building blocks of brillin microbrils.

Gremlin-1 and brillin-2 are overexpressed in the mesothelioma tumor tissue and colocalize in vivo

To analyze whether gremlin-1 and brillins are coexpressed in vivo, tissue biopsies from mesothelioma patients included in this study were stained with specic antibodies. We had previously shown low levels of gremlin expression in the normal lung.16 Here, we detected low expression of gremlin in normal mesothelial cells (Figure 2a). Gremlin-1 staining was, however, detected in non-malignant, reactive mesothelial cells in control samples (pneumothorax). Intense gremlin-1 immunoreactivity was observed in all mesothelioma samples (n 6). Fibrillin-2 immu

noreactivity was shown in a strikingly similar staining pattern as observed for gremlin-1 in these tumor samples, suggesting concomitant upregulation of these developmental genes in mesothelioma. Some brillin-2 staining was detectable also in non-malignant reactive mesothelium. Calretinin and WT1 (Wilms tumor 1) are diagnostic markers for mesothelioma and were used here to identify the tumor tissue. Analyses of serial tissue sections suggested that gremlin-1 and brillin-2 staining localizes to calretinin-positive and -negative tumor areas (Figure 2b). Some WT-1 positivity, however, was observed in calretinin-negative areas, which represent sarcomatoid part of the tumor with more diffuse growth properties. Fibrillin-1 staining was negative in 5/6 mesothelioma and in control pleura samples. Only one

mesothelioma sample showed brillin-1 immunoreactivity, which was specically detected in stromal-like areas (not shown).

We analyzed colocalization of gremlin-1 with brillin-2 in tumor tissue using the proximity ligation assay. Intensive colocalization signals were observed for gremlin-1 and brillin-2 throughout the tumor tissues, suggesting that they localize to similar structures in vivo (Figure 3).

Primary human mesothelioma cells express high levels of gremlin-1 and brillin-2

Primary cells were cultured from mesothelioma patients pleural effusion samples (JP1-5). Cells were characterized by immunouorescence staining using mesothelial markers calretinin and cytokeratin (CK)-7 as well as vimentin, which is commonly expressed by tumor cells. Majority of the cells were calretininand/or CK-7 positive and stained also for vimentin (Figure 4a). This suggests that the cells, which were able to proliferate, were mainly primary tumor cells. Gremlin-1 mRNA expression levels were high in these primary cells compared with Met5A cells, which are immortalized but non-tumorigenic mesothelial cells (Figure 4b). Further, the mRNA expression levels of brillin-1 and -2 were high in these cells (Figure 4b). The results show that primary mesothelioma cells can be cultured from pleural effusion samples and that the cells retain the phenotypic expression of these developmental genes.

Gremlin-1 is known to inhibit the functions of BMP-2, -4 and to some extent also BMP-7.7,8 Therefore, the expression levels of these BMP isoforms were analyzed in primary mesothelioma cells.

Figure 2. Overexpression of gremlin-1 and brillin-2 in mesothelioma. (a) Immunohistochemical staining of mesothelioma and control pleura samples using calretinin, gremlin-1, brillin-1 and brillin-2 antibodies. Hematoxylin and eosin (HE) staining is shown on the left. Abundant gremlin-1 and brillin-2 immunoreactivity is observed in both calretinin-negative and -positive tumor areas. Normal mesothelium (pleura, left panel) shows very low levels of staining, whereas reactive normal mesothelium (pleura, right panel) shows faint brillin-2 and moderate gremlin-1 staining. (b) Immunohistochemical staining of serial mesothelioma sections suggests similar staining patterns for gremlin-1 and brillin-2. Staining was observed also in calretinin-negative stromal-like areas, which contained isolated Wilms tumor protein (WT1)-positive tumor cells (white arrows).

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Figure 3. Colocalization analyses of gremlin-1 with brillin-2 in mesothelioma tumor tissue. Proximity ligation assay was used to detect colocalization of gremlin-1 with brillin-2 in mesothelioma tumor tissue. Positive colocalization signals (red dots) were abundant throughout the tumor tissue. Negative control with gremlin antibody plus mouse isotype control is shown on the upper right panel.

Interestingly, abundant expression of BMP-2 was observed (Figure 4b), whereas BMP-4 levels were comparable and BMP-7 levels only slightly higher compared with Met5A cells (not shown).

The expression patterns of gremlin-1, brillins and BMP-2 were also analyzed in four established mesothelioma cell lines (211H, H28, H2452 and H2052). Only one of these cell lines, H2052, resembled primary mesothelioma cells and expressed high levels of gremlin-1, brillin-2 and BMP-2 (Figure 4c). Therefore, this cell line was chosen for further mechanistic studies.

Gremlin-1 associates with brillin-1 in cultured mesothelioma cells Mesothelioma cells were cultured for 1 week and then analyzed by immunouorescence staining using antibodies specic for gremlin-1 or brillins. Although expressed at the mRNA level, brillin-2 protein was not detected in any of the cultured mesothelioma cells analyzed at this time point (not shown), which may reect late extracellular matrix (ECM) deposition in vitro. Primary mesothelioma cells and H2052 cells in vitro also expressed brillin-1 mRNA, and this was reected in the staining pattern showing brillar staining for brillin-1 and gremlin-1 (Figure 5a). No staining was observed in H2452 cells. Double immunouorescence labeling suggested colocalization and targeting of gremlin-1 into brillin-1 containing microbrils in vitro. H2052 cells transfected with brillin-1-specic siRNA showed reduced mRNA expression levels (Figure 5b) and signicantly reduced brillin-1 staining, indicating efcient silencing of protein expression (Figure 5c). This led to reduced deposition of gremlin into brillar structures suggesting that extracellular targeting of gremlin is dependent on brillins.

Gremlin-1 silencing severely impairs mesothelioma cell growth and survival

To investigate the role of gremlin-1 in mesothelioma cell growth, H2052 cells were transfected with gremlin-specic siRNAs.

Efcient silencing of gremlin-1 expression was observed at mRNA and protein levels (Figures 6a and b). Cell proliferation was assayed by counting cells 15 days after transfection. Two independent siRNAs targeted against gremlin-1 were observed to signicantly decrease the number of cells at all time points (Figure 6c). This suggests that H2052 cell proliferation is dependent on gremlin expression.

To determine whether lack of gremlin induces apoptosis, H2052 cells were cultured on coverslips, transfected with siRNAs and analyzed with TUNEL assay 3 days after transfection (Figure 6d). Hardly any TUNEL-positive cells were detected in control or gremlin siRNA-transfected cells. DAPI staining also showed intact nuclei in both groups. These results suggest that although lack of gremlin impaired cell proliferation, it did not induce apoptosis.

Cellular signaling pathway activities are altered in gremlin-1-silenced cells

Changes in BMP signaling activity were analyzed. Gremlin-1 silencing increased BMP-dependent reporter activity in H2052 cells (Figure 7a) as well as the expression of a BMP target gene Id1 (inhibitor of differentiation/DNA binding 1, Figure 7b), suggesting that endogenous gremlin-1 regulates BMP activity negatively. Further, Id1 expression was found signicantly reduced in primary mesothelioma cells compared with Met5A cells (Figure 7c).

To further investigate cellular signaling pathways regulated by gremlin, we analyzed alteration in phospho-kinase levels using a commercial array (see Materials and methods). H2052 cells were found to have high basal level of phospho-Akt (S473), which was not altered in gremlin-1-silenced cells (Figures 7df). However, phosphorylation of the Akt substrate and regulator of mTOR signaling, PRAS40 (T246), was increased. Erk1/2 phosphorylation was also signicantly increased in gremlin-1-silenced cells (g). Further, increased levels of p53 phosphorylation (S46 and S392)

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Figure 4. Primary mesothelioma cells express high levels of gremlin-1. Primary mesothelioma cells (JP cells) were isolated from pleural effusion samples. (a) Immunouorescence staining suggests that the cells were positive for the mesothelial marker calretinin. Co-staining with cytokeratine (CK)-7 and vimentin antibodies suggests that the tumor cells co-express these marker proteins. (b) Primary mesothelioma cells were analyzed for gremlin-1, brillin-1 and -2 and BMP-2 mRNA expression by quantitative RTPCR. The levels were normalized to the expression levels of TATA-binding protein and are expressed relative to the expression levels in Met5A cells (immortalized, non-tumorigenic mesothelial cells), which were set to 1. (c) Established mesothelioma cell lines were analyzed for gremlin-1, brillin-1 and -2 and BMP-2 mRNA expression by quantitative RTPCR. The error bars represent s.d. (n 2).

were reected in increased mRNA expression levels of p21 (Cip1/ Waf1) in H2052 cells (Figure 7h). In agreement, overexpression of gremlin-1 in H2052 and H28 cells decreased p21 expression levels (Figure 7i).

Gremlin-1 expression is associated with EMT phenotype and chemoresistance

Gremlin-1 expression has been linked to EMT processes. Therefore, we determined whether mesothelioma cells with high gremlin levels express transcription factors snail and slug, which are transcriptional repressors of E-cadherin and induce EMT.17

Whereas the mRNA expression levels of snail were comparable in mesothelioma cells and Met5A cells, slug expression levels were high in primary mesothelioma cells (Figure 8a). Slug expression in mesothelioma tumor samples was analyzed using immunohisto-chemical staining (Figures 8b and c). All of the analyzed tumor samples (n 6) were positive for slug. Mesothelioma tumors

exhibited mostly diffuse and granular cytoplasmic staining, but also nuclear staining was detected in certain tumor areas. Slug staining localized to gremlin-positive areas and was detected also in calretinin-negative areas.

Similar to primary mesothelioma cells H2052 cells, which express high levels of gremlin-1, exhibited high slug expression (Figure 8d). Conversely, Met5A and H2452 cells showed very low levels of slug mRNA expression consistent with low gremlin-1 expression levels. Slug protein levels also showed a dramatic difference between H2052 and H2452 cells (Figure 8d). Silencing of gremlin-1 by siRNA transfection signicantly reduced slug mRNA and protein expressions in H2052 cells (Figure 8e and i). Similar results were also obtained with primary mesothelioma cells (Figure 8f). Further, overexpression of gremlin-1 in H28 cells led to increased slug mRNA expression (Figure 8g). The mRNA and especially protein expression levels of mesenchymal proteins, N-cadherin, vimentin and a-smooth muscle actin were signicantly downregulated in gremlin-1-silenced H2052 cells (Figures

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Figure 5. Gremlin-1 and brillin-1 co-localize in mesothelioma cell ECM in vitro. (a) Mesothelioma cell lines (H2052 and H2452) and primary mesothelioma cells (JP5) were co-stained for gremlin-1 and brillin-1 and analyzed using immunouorescence microscopy. A brillar staining pattern was observed in H2052 and JP5 cells. (b) In control (ctrl) and brillin-1 siRNA-transfected H2052 cells, the mRNA expression levels of gremlin-1 and brillin-1 were analyzed by quantitative RT-PCR. The levels were normalized to the expression levels of TATA-binding protein and are expressed relative to each control, which was set to 1. The error bars represent s.d. (n 2). (c) Double-immunouorescence staining of

siRNA-treated H2052 cells with gremlin-1 and brillin-1 antibodies. Note the lack of brillar gremlin-1 staining in brillin-1-silenced cells.

8h and i). Fibrillin-1 and -2 mRNA expression levels were also signicantly reduced (Figure 8h), suggesting that gremlin-1 maintains the expression of microbrillar genes. Further, bronectin expression was downregulated (Figure 8h), and the epithelial marker E-cadherin was upregulated (Figure 8j). These results suggest that EMT-related alterations are reversed in gremlin-1-silenced cells.

Slug expression and EMT-phenotype are also associated with tumor chemoresistance.1821 The importance of high gremlin and slug expression to sensitivity to paclitaxel-mediated mesothelioma cell death was analyzed using the MTT proliferation/viability assay. H2052 cells, which express high levels of gremlin-1 and slug, were

found more resistant to paclitaxel than H2452 cells (Figure 8k). Next, control and gremlin-1 siRNA-transfected H2052 cells were treated with paclitaxel or pemetrexed, and cell numbers were quantied 3 days after transfection. Treatment with paclitaxel or pemetrexed in combination with gremlin-1 siRNA led to efcient cell death (Figure 8l).

DISCUSSIONGremlin-1-mediated inhibition of BMP-signaling is important for normal development. Mice lacking gremlin-1 die before birth because of severe defects in kidney and lung development.10 In

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Figure 6. Gremlin-1 silencing impairs H2052 mesothelioma cell proliferation. (a) The efciency of gremlin-1 siRNA silencing was analyzed with quantitative RT-PCR as in gure 5. The error bars represent s.d. (n 4). (b) Double immunouorescence staining of siRNA-treated H2052 cells

with gremlin-1 and brillin-1 antibodies. Note the lack of brillar gremlin-1 staining, which is suggestive of efcient silencing using gremlin siRNA3. (c) H2052 cell proliferation was analyzed by counting cells 15 days after siRNA transfection. Both gremlin siRNAs reduced dramatically the number of cells 35 days after transfection (left panel, a representative experiment is shown). Same effect was seen also at earlier time points (right panel). The error bars represent s.d. (n 3, *P 0.05). (d) Silencing gremlin-1 by siRNA3 did not induce apoptosis as

measured using TUNEL staining. DNase I-treated cells (control) show positive staining. Nuclei are stained with DAPI.

adult tissues including lung gremlin-1 expression is low. In idiopathic pulmonary brosis , gremlin-1 is highly upregulated in the lung parenchyma, where it contributes to idiopathic pulmonary brosis pathogenesis by blocking BMP-mediated signals.16 Gremlin-1 has been found to be upregulated also in many carcinoma tissues as well as in mesothelioma.12,13,22 The

role of gremlin-1 in malignant tissue appears complex and is likely mediated both by BMP-dependent and -independent

functions. There are reports suggesting tumor-specic gremlin-1 expression,12 while other reports suggest expression in cancer-associated stromal cells.13 Here, we nd high gremlin immunoreactivity in the mesothelioma tumor tissue. Analyses of serial mesothelioma tissue sections suggested that gremlin-1 can localize to calretinin-positive, epithelial-appearing tumor areas and also to aberrant looking and stromal-like calretinin-negative areas. These tissue areas contained some WT1-positive tumor cells.

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Figure 7. Cellular signaling in gremlin-1-silenced cells. (a) BMP-dependent reporter activity was analyzed in control and gremlin-1 siRNA-treated H2052 cells. The levels are expressed relative to control, which was set to 1. The error bars represent s.d. (n 4, *Po0.01). (b) Id1 mRNA

expression levels were analyzed by quantitative RTPCR. The levels are expressed relative to control. n 3, *Po0.05. (c) Id1 mRNA expression

levels in primary mesothelioma cells. The levels are expressed relative to the expression levels in Met5A cells. (d) Cell lysates from control and gremlin-1 siRNA-treated H2052 cells were analyzed using a commercial phospho-protein array. (e) Quantication of alterations in the amounts of phospho-proteins (n 2). Changes were also veried by immunoblotting analyses of Akt (f) and ERK (g) pathway proteins. Molecular

weight markers are indicated on the left. Expression levels of p21 (Cip1/Waf1), a p53 target gene, were analyzed in gremlin-1 siRNA-treated (h) and gremlin-1-transfected cells (i) by quantitative RT-PCR. The levels are expressed relative to control, which was set to 1. n 3, *Po0.05. I,

inset. Gremlin-1 overexpression in H2052 and H28 cells was veried by immunoblotting using an antibody against the V5 tag.

Our results suggest that gremlin-1 localizes mainly in and around mesothelioma tumor cells.

Using a sophisticated large-scale protein interaction screen, we were able to identify brillin-2 as a novel gremlin-1 interacting protein. The interaction was also veried using a cell surface plasmon resonance technology. Fibrillin-2 is a developmental gene, which is found in adult vasculature and is upregulated in idiopathic pulmonary brosis, scleroderma and during skin wound healing.23,24 Idiopathic pulmonary brosis and cancer pathogenesis have common features including aberrant activation of mediators of tissue repair processes.25 Our discovery that high brillin-2 immunoreactivity is associated with mesothelioma tumor tissue is a novel nding and supports this idea. Fibrillins (brillin-1 and -2) are large extracellular glycoproteins, which form heteromeric brillar structures, the so called brillin microbrils, which are essential for elastin assembly.26 Fibrillins also regulate growth factor signaling by sequestering TGF-b complexes as well as BMP-isoforms into extracellular matrix structures.27 Gremlin-1 can bind to and inhibit the functions of BMP-2, -4 and -7, all of which have been shown to associate with brillins.9 Here, we nd

that gremlin-1 can bind to brillin-1 and -2 in vitro and colocalizes with brillin-2 in mesothelioma tumor tissue in vivo. Conceptually this is a new nding, spatially localizing a BMP inhibitor molecule with its target proteins through brillin microbrils. High levels of brillin-2 are likely to contribute to tumor cell behavior through aberrant extracellular matrix structure and regulation of gremlin-1 function.

Primary mesothelioma cells were cultured from patients pleural effusion samples and analyzed after few passages of culture. Immunouorescence microscopy analyses indicated that the cells, which were able to grow, represented mostly tumor cells. Gremlin-1 was expressed at mRNA and protein levels and brillin-2 at mRNA level by these primary cells, suggesting that they retain the pathological expression pattern of these genes. Interestingly, brillin-1 but not brillin-2 protein was detected in the matrix structures after 1 week culture of the primary cells. This likely reects the temporal production of ECM proteins and remodeling of matrix structures during in vitro cell cultures.28,29 Fibrillin-2 is likely assembled late during this process. In agreement with the protein interaction studies, gremlin-1 localized to brillin-1-

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Figure 8. Gremlin-1 regulates slug expression and EMT phenotype. (a) Primary human mesothelioma cells (JP) were analyzed for snail and slug mRNA expression by quantitative RT-PCR. The levels were normalized to the expression levels of TATA-binding protein and are expressed relative to the expression levels in Met5A cells, which were set to 1. (b, c) Immunohistochemical staining of a mesothelioma section using slug antibodies shows mostly diffuse and granular cytoplasmic staining in the tumor tissue. Nuclear staining was also observed in certain tumor areas (b, right panel). (c) Analyses of serial mesothelioma tissue sections suggested that slug and gremlin staining colocalized in the same tumor areas. (d) Slug mRNA expression levels in H2052 and H2452 cells were analyzed by quantitative RTPCR. The levels are expressed relative to H2452 expression levels, which were set to 1. The error bars represent s.d. (n 5). INSET: immunoblotting analyses of slug. Tubulin is

used as a loading control. Molecular weight markers are indicated on the left. (eg) Slug mRNA expression levels were analyzed in control and gremlin-1 siRNA-treated H2052 and JP5 cells or H28 cells transfected with gremlin-1 (grem1) or control vector (mock). The levels are expressed relative to control, which was set to 1. The error bars represent s.d. (JP5, n 3, *Po0.05; H2052, n 4, *Po0.01; H28, n 3, *P o0.05).

(h) N-cadherin, vimentin, a-SMA, bronectin and brillin mRNA expression levels were analyzed in control and gremlin siRNA-treated H2052 cells by quantitative RT-PCR. The error bars represent s.d. (nX3, *Po0.05). (i) Immunoblotting analyses of slug, N-cadherin, vimentin and a-SMA in control and gremlin-1 siRNA-treated H2052 cells. Tubulin is used as a loading control. (j) E-cadherin mRNA expression levels were analyzed in control and gremlin siRNA-treated H2052 cells by quantitative RTPCR. Error bars represent s.d. (n 3, *Po0.05). (k) H2052 and

H2452 cells were treated with the indicated concentrations of paclitaxel after which cell proliferation/viability was analyzed with MTT assay (n 4, *Po0.05). (l) Quantication of cell numbers after combined treatment with control or gremlin-1 siRNA and paclitaxel (0.1 n

M, 24 h) or

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pemetrexed (1 mM, 48 h). Cell numbers were counted 3 days after transfection (nX4, *P 0.002).

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Figure 9. A model for the functions of gremlin-1 and brillin-2 in mesothelioma. Gremlin-1 induces proliferation and epithelial-tomesenchymal transition (EMT) in mesothelioma cells. EMT is mediated by induction of the transcription factor slug and leads to chemoresistance and acquisition of invasive properties. Gremlin-1 is targeted to the tumor microenvironment through binding to brillin-2 microbrils.

containing structures in mesothelioma cell ECM. Gene expression analyses suggested that compared with immortalized but nontumorigenic mesothelial cells (Met5A), primary mesothelioma cells also expressed signicantly higher levels of BMP-2. This is an interesting nding as BMP-2 has been linked to tumor invasion and metastasis.3032 Future studies will reveal whether BMP-2 is functional in the presence of high levels of its inhibitor protein.

We used the knowledge of gene expression patterns of primary mesothelioma cells to nd a representative established mesothelioma cell line for mechanistic studies. H2052 cells expressed high levels of gremlin-1, brillin-2 and BMP-2 similar to all of the primary cells. Gremlin-1 silencing in these cells reduced cell proliferation dramatically. This is in agreement with previous studies suggesting that gremlin-1 can induce proliferation of cancer cells.3335 Interestingly, we observed gremlin-1 immunoreactivity also in reactive mesothelium, which associates gremlin-1 expression to a proliferative phenotype even in non-malignant mesothelial cells. Gremlin-1 expression has been linked to mesenchymal cells surrounding stem cell niches, which support proliferation of epithelial cells through blocking BMP activities.13,36

Mesenchymal stem cells also express high levels of gremlin-1 (GeneSapiens database37). Our data suggest a model in which cancer cells can acquire the ability to produce high levels of gremlin-1, which is targeted to the tumor microenvironment through brillin binding (see Figure 9).

Signaling pathways linked to the regulation of cell survival, apoptosis and autophagy were altered in gremlin-1-silenced H2052 cells. Similar to our ndings, negative regulation of Erk1/2 phosphorylation by gremlin-1 has been previously found in osteosarcoma cells;38 however, gremlin overexpression in Saos-2 cell was found to reduce proliferation. We observed increased p53 phosphorylation and expression of its target gene, p21, in gremlin-1-silenced cells, suggesting a link to reduced cell survival. Further, the phosphorylation of the Akt substrate PRAS40 was signicantly increased. Although PRAS40 has been linked to diabetes and cancer, its pathological functions are not well understood.39 Interestingly, gremlin-1 has been shown to bind directly to a 14-3-3 protein,12

which in turn binds and regulates the PRAS40 and other Akt/mTOR

pathway proteins. Future studies will reveal the mechanistic details how cellular and secreted gremlin-1 regulates these pathways.

We and others have previously linked gremlin-1 expression to EMT processes.16,40,41 In cancer cells, markers of EMT often

suggest more advanced disease, invasive capacity and chemoresistance.21,42 We found that the transcription factor

slug, a master regulator of EMT, was upregulated in mesothelioma cells concurrently with gremlin-1. Silencing of gremlin-1 expression led to reduced slug mRNA and protein levels. Further, mesenchymal markers of EMT, vimentin, a-smooth muscle actin, bronectin and N-cadherin were downregulated in gremlin-1-silenced cells, whereas epithelial marker E-cadherin was upregulated. These results are in agreement with previous studies linking the expression of EMT-related transcription factors to mesothelioma43,44 and provide evidence that gremlin

functions as a regulator of these transitional processes. EMT and slug expression have also been suggested to be involved in regulating cancer stemness as well as chemoresistance.18,20,42

In agreement, we observed that high gremlin-1 and slug expressions in mesothelioma cells were associated with reduced sensitivity to paclitaxel-mediated cell death. Combined treatment with paclitaxel and gremlin-1 siRNA led to efcient cell death in vitro. Similar results were obtained with pemetrexed, which is a relatively new cancer drug used in the treatment of mesothelioma in combination with platinum compounds.45 This

indicates that blocking gremlin-1 function may be benecial in mesothelioma especially when combined with chemotherapy.

Mesothelioma is an aggressive tumor with limited treatment options. Further, an increase is expected in the incidence of mesothelioma,3,4 which sets an urgent need for new drug target molecules. Although the number of patient tissues analyzed in our study was relatively small (n 6), the pathological expression of

gremlin-1 was a clear concept. It is also reinforced by other studies suggesting a role for gremlin-1 in cancer cell proliferation.12,13,33,34

Gremlin-1 has also BMP-independent angiogenic functions,46

which may well contribute to tumor progression. Blocking gremlin-1 function in mesothelioma might offer means to ght the chemoresistance of this tumor.

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MATERIALS AND METHODSAntibodiesAntibodies used in the study are summarized in Table 2.

Patients and tissues specimenA statement for the use of human tissue and pleural effusion materials was received from the Ethics Committee of the Helsinki University Hospital, Helsinki, Finland (number 308/13/0301/2010). All patients gave informed consent to participate in the study. Tissue biopsies and pleural effusion samples were obtained from patients undergoing diagnostic procedures and had a clinical and/or radiological suspicion of malignant mesothelioma. All patients included in the study (n 7) were later diagnosed to

have mesothelioma (Table 3). Control pleural samples were obtained from two male smokers who were operated for pneumothorax.

Cell culture and transient transfectionImmortalized normal mesothelial cells (Met5A),47 and mesothelioma cell lines (211H, H28, H2452 and H2052) were from ATCC. Primary mesothelioma cells (named JP cells) were acquired from pleural effusion samples from patients suffering from malignant mesothelioma. Cells were centrifuged, washed and seeded on plastic culture dishes in RPMI-1640 medium (Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma) and antibiotics. The medium was changed every 34 days. Cells were passaged 23 times before immunouorescence analysis of mesothelioma marker proteins was performed. Cells were also cultured for 67 days for RNA isolation and gene expression analyses. For further experiments JP5 cells were cultured and used between passages 37. Transfection of cells was carried out using siRNAs for gremlin, brillin-1 or a negative control (Life Technologies, Paisley, UK) as described.48 The BMP

reporter construct (Bre2-luc, kindly provided by Dr Peter ten Dijke, Leiden University Medical Center, the Netherlands) was transfected into H2052 cells to assess BMP-dependent signaling activity as described.49

Expression constructs and stable transfectionC-terminal gremlin-1 construct was generated by Gateway-cloning, the gremlin-1 construct from the Human Orfeome Collection (Thermo Scientic, FL, USA) to a pTO-SHc-GW-FRT plasmid with c-terminal Streptag III and human inuenza hemagglutinin (HA)-tag.14 Flip-In human embryonic kidney (HEK) 293 cells (Life Technologies) were grown as recommended. Stable transfection of cells was carried out as described.50

After 2 weeks of culture, hygromycin B-resistant clones were tested for tetracycline (Sigma)-inducible gremlin-1 expression (see Figure 1b).

Afnity puricationTransfected Flip-In HEK293 cells were stimulated with tetracycline (25 ng/

ml) to induce gremlin-1 expression. After a 24-h stimulation, cells were lysed in HNN-lysis buffer (50 mM HEPES pH 8, 150 mM NaCl, 50 mM NaF, 1 mM PMSF, 1.5 mM Na-vanadate, 0.5% NP-40 and protease inhibitors (Sigma)) followed by double afnity purication as described.50 Briey, cell lysates were passed through Strep-Tactin columns (IBA GmbH) followed by elution of bound proteins with 2.5 mM biotin. Next, the eluate was immunoprecipitated using anti-HA agarose (Sigma). Proteins were nally eluted using 0.2 M glycine and neutralized.

Protein digestionThe total protein amount from the elution fractions was precipitated using trichloroacetic acid (TCA). First, TCA was added to the elution fractions to nal concentration of 25% followed by incubation for 30 min on ice. The precipitates were centrifuged, washed once with ice cold 0.1 M HCl in acetone and once with acetone. The pellets were then allowed to dry. For trypsin digestion the precipitated proteins were dissolved in 50 mM

ammonium bicarbonate, pH 8.9, containing 0.1% Rapigest detergent (Waters, Milford, MA, USA). Proteins were reduced using DTT for 30 min at 60 1C followed by alkylation using iodoacetamide for 30 min at room temperature in dark. Proteins were digested with trypsin (Promega, Madison, WI, USA) for 18 h at 37 1C, and then the Rapigest detergent was hydrolyzed with HCl for 45 min at 37 1C. Peptides were puried with PepClean C18 Spin Columns (Thermo Fisher, Rockford, IL, USA) according to the manufacturers protocol. Finally, the puried peptides were dissolved in 0.1% formic acid for MS analysis.

Table 3. Mesothelioma patient characteristics

Patient Type Location Age Gender IHC Cells

JP1 Epithelial Pleura 53 Male

JP2 Biphasic Pleura 71 Male

JP3 Epithelial Pleura 63 Male

JP4 Biphasic Peritoneum 72 Female

JP5 Epithelial Pleura 69 Female

JP6 Biphasic Pleura 71 Female

JP7 Epithelial Pleura 61 Male Tissues analyzed by immunohistochemistry (IHC) and pleural effusion samples obtained for cell culture (cells) are indicated.

Table 2. List of antibodies used in the study

Name Source Application Supplier

calretinin mouse/DAK-Calret1 IF, IHC DAKO, Glostrup, Denmark, Germany cytokeratin-7 rabbit /EP16204 IF Novus Biologicals, Littleton, CO, USA vimentin mouse/V9 IF, IB Santa Cruz, Santa Cruz, CA, USA N-cadherin mouse/32 IB BD Biosciences, Franklin Lakes, NJ, USA a-SMA mouse/1A4 IB Sigma, St Louis, MO, USA

HA mouse/16B12 IB Covance, Princeton, NJ, USA brillin-1 mouse/2499 IF Millipore, Billerica, MA, USA brillin-1 rabbit/ pAb9543 IHC, IF Professor Lynn Sakaibrillin-2 mouse/48 IHC, PLA Milliporegremlin-1 rabbit pAb IHC Abcam, Cambridge, UK gremlin-1 goat pAb IHC, PLA Santa Cruzgremlin-1 rabbit pAb IF Santa CruzSlug rabbit pAb IB, IHC SigmaWT1 mouse/WT49 IHC Novocastra, Newcastle, UK p-Akt (S473) rabbit pAb IB Cell Signaling, Danvers, MA, USA p-Akt (T308) rabbit pAb IB Cell SignalingAkt rabbit pAb IB Cell Signalingp-Erk1/2 rabbit mAb/D13.14.4E IB Cell SignalingErk1 mouse/3A7 IB Cell SignalingV5 Mouse mAb IB Invitrogen

Abbreviations: IB, immunoblotting; IF, immunouorescence; IHC, immunohistochemistry; PLA, proximity ligation assay.

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LC-MS/MS analysis

Mass spectrometric analysis was performed using nanoAcquity UPLC

liquid chromatography system on-line with a Waters Synapt G2 mass spectrometer (Waters S.A.S., Saint-Quentin, France). Waters nanoAcquity UPLC Trap Column (Symmetry C18, 180 mm 20 mm, 5 mm) was used as

trapping and Waters nanoAcquity UPLC Column (BEH130 C18, 75 mm 150

mm, 1.7 mm) as analytical column. Four microliters of sample was injected and run using 90 min gradient from 3 to 40% mobile phase B (0.1% formic acid in acetonitrile). 0.1% formic acid in water was used as mobile phase A.

Data were collected with data dependent acquisition manner collecting eight fragmentation spectra simultaneously. Switch limit from MS to MS/ MS was set to peak intensity of 200. Fragmentation data for detected peaks were collected for 5 s and then excluded from fragmentation for 120 s. Scan time for both MS and MS/MS was one second. No lockmass correction was used.

The raw data were processed with Mascot Distiller software (version2.3.1.0, Matrix Science, London, UK), and database search was performed using Mascot search engine (version 2.2.04) against Swiss-Prot human database (dated 16.6.2010). MudPIT scoring and Ion score cutoff limit of 20 were used to identify peptides. Require bold red- specication was used to limit the results to those proteins that have at least one unique peptide. Carbamidomethylated cysteine was set as xed modication and oxidation of methionine as variable modication. Peptide mass tolerance for search was 0.2 Da and 0.1 Da for fragment ion search. A maximum of two missed trypsin cleavages was allowed. The Mascot Distiller processed data were also searched using GPM X!Tandem search engine51 using same parameters.

Expression and purication of recombinant gremlin-1The expression construct pGremlin-1_V5/HIS in pEF-IRES-P contains cDNA encoding human gremlin-1 with C-terminal tags (V5 and 6HIS). H2052 and H28 cells were transiently transfected with this construct or the expression vector using Fugene HD transfection reagent (Promega). CHO cells were stably transfected, and gremlin-containing cell culture supernatants were produced as described.52 The media was then dialyzed against 50 mM

sodium phosphate buffer, pH 7.2 and 1 M NaCl (buffer A) and loaded on a 1-ml HiTrap column (GE Health Care, Waukesha, WI, USA) previously charged with CoCl2 and equilibrated with buffer A. The loaded column was washed with 40 column volumes of buffer A and subjected to a fast protein liquid chromatography (GEHC) gradient run starting with buffer A containing 0100% buffer B (buffer A with 250 mM imidazole). Fractions were analyzed using SDSPAGE for their purity and molecular mass, and imidazole in these fractions was removed by dialysis against buffer A. For further concentration and purication, gremlin-1-containing fractions were dialyzed against TBS and subjected to afnity chromatography using a 1-ml HiTrap heparin column (GEHC). Bound gremlin-1 was eluted in a 01 M

NaCl gradient.

Surface plasmon resonanceBinding analyses were performed using a BIAcore2000 (GEHC). 2000 Rus of gremlin-1 were covalently coupled to carboxymethyldextran hydrogel 500 M sensor chips (XanTec, Dsseldorf, Germany) using the amine coupling kit following the manufacturers instructions (GEHC). Binding assays with N-terminal brillin-1 (rF11) and -2 peptides (rF86) as analytes were performed as previously described.9 Kinetic constants were calculated by nonlinear tting of association and dissociation curves (BIAevaluation 4.1 software).

Immunohistochemical staining and proximity ligation assay Immunohistochemical staining of tumor tissues and proximity ligation assay were performed as described.24 Images were captured with Nikon DS-Fi1 or with Axio Imager with ApoTome (Zeiss, Gttingen, Germany) or with using Axio Vison 4.8 software (Zeiss).

SDSPAGE and immunoblottingCells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.2% sodium deoxycholate) containing protease inhibitors (Roche, Mannheim, Germany) for 15 min on ice. Protein concentrations were measured using a BCA protein assay Kit (Pierce, Rockfors, IL, USA). SDSPAGE and immunoblotting were performed as described.53

RNA isolation and gene expression analysesIsolation of total cellular RNA, reverse transcription and quantitative real-time PCR were carried out as described.53 The relative gene expression differences were calculated with the comparative delta delta cycle threshold (DDCT) method, and the results have been expressed as mRNA expression levels normalized to the levels of a gene with a constant expression (TATA-binding protein).

Immunouorescence analyses and microscopyCells were grown on glass coverslips for the indicated times and xed with 4% paraformaldehyde in PBS at room temperature for 10 min or with ice cold methanol at 20 1C for 20 min. Immunouorescence staining was

carried out as described.53 Images were captured with a Axioplan 2 epiuorescence microscope (Zeiss) and AxioCam HRm 14-bit grayscale CCD camera Axiovision 4.6 software (Zeiss).

Human phospho-kinase arrayControl and gremlin-1 siRNA-treated cells were lysed, and alterations in phospho-kinase levels were analyzed using a Proteome proler array (ARY003b, R&D Systems, Gaitherburg, MD, USA) according to the manufacturers instructions. Quantity One version 4.6 (BioRad, Hercules, CA, USA) was used for quantication. The kinase array was performed twice, and the results are expressed as average of the two experiments.

Measurements of cell viability and apoptosisCell proliferation/viability was assessed using MTT assay (R&D Systems).

Briey, cells were seeded on 96-well plates (10 000/well) and were treated the next day with different concentrations of paclitaxel (Hospira, Lake Forest, IL, USA). The metabolic activity of cells was measured after 48 h according to the manufacturers instructions. Apoptosis was assayed using a TUNEL technology using In Situ cell death detection kit (Roche). Cells were seeded on glass coverslips, cultured overnight and transfected with siRNAs. Cells were then xed and stained, according to the manufacturers instructions, 3 days after transfection.

Statistical analysesData were analyzed using PASW Statistics 18 program for Windows (SPSS,

Chicago, IL, USA). Statistical difference between two independent groups was evaluated using nonparametric MannWhitney U-test. Statistical difference between more than two independent groups was evaluated using nonparametric KruskalWallis test. A P-value of o0.05 was considered statistically signicant.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGEMENTS

We thank Eva Sutinen for coordinating patient sample collection and performing immunohistochemical staining experiments, Arja Pasternack for CHO cell transfection and production of conditioned media, Sami Starast for technical assistance, Professor Jorma Keski-Oja for discussions, Professor Lynn Sakai (Oregon Health and Science University, Portland, OR) for the brillin-1 antibody and brillin-1 and -2 peptides, and Biomedicum Imaging Unit for imaging support. This work was supported by Academy of Finland, Sigrid Juslius Foundation, Jalmari and Rauha Ahokas Foundation, Magnus Ehrnrooth Foundation, Finnish Cultural Foundation, Foundation of the Finnish Anti-Tuberculosis Association, Deutsche Forschungsgemeinschaft (SFB829, project B12 to GS).

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