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PIGMENT CELL & MELANOMA Research ADAM10 correlates with uveal melanoma metastasis and promotes in vitro invasion Rosaria Gangemi, Adriana Amaro, Alice Gino, Gaia Barisione, Marina Fabbi, Ulrich Pfeffer, Antonella Brizzolara, Paola Queirolo, Sandra Salvi, Simona Boccardo, Marina Gualco, Francesco Spagnolo, Martine J. Jager, Carlo Mosci, Armando Rossello and Silvano Ferrini

DOI: 10.1111/pcmr.12306 Volume 27, Issue 6, Pages 1138–1148 If you wish to order reprints of this article, please see the guidelines here Supporting Information for this article is freely available here EMAIL ALERTS Receive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here

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ORIGINAL ARTICLE

Pigment Cell Melanoma Res. 27; 1138–1148

ADAM10 correlates with uveal melanoma metastasis and promotes in vitro invasion Rosaria Gangemi1, Adriana Amaro1, Alice Gino1, Gaia Barisione1, Marina Fabbi1, Ulrich Pfeffer1, Antonella Brizzolara1, Paola Queirolo1, Sandra Salvi1, Simona Boccardo1, Marina Gualco1, Francesco Spagnolo1, Martine J. Jager2, Carlo Mosci3, Armando Rossello4 and Silvano Ferrini1 1 IRCCS A.O.U. San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 2 Department of Ophthalmology, Leiden University Medical Center (LUMC), Leiden, The Netherlands 3 Ocular Oncology Center, Galliera Hospital, Genoa, Italy 4 Department of Pharmacy, University of Pisa, Pisa, Italy CORRESPONDENCE: Silvano Ferrini, e-mails: [email protected]; [email protected]; [email protected]

KEYWORDS ADAM10/ADAM17/uveal c-Met/invasion/gene expression

melanoma/

PUBLICATION DATA Received 4 March 2014, revised and accepted for publication 12 August 2014, published online 2014 Aug 14 doi: 10.1111/pcmr.12306

Summary Uveal melanoma (UM) is a rare ocular tumor that may lead to deadly metastases in 50% of patients. A disintegrin and metalloproteinase (ADAM)10, ADAM17, and the HGF-receptor c-Met support invasiveness in different tumors. Here, we report that high ADAM10, MET, and, to a lesser extent, ADAM17 gene expression correlates with poor progression-free survival in UM patients (hazard ratio 2.7, 2.6, and 1.9, respectively). About 60% of primary UM expresses c-Met and/or ADAM10 proteins. Four UM cell lines display high levels of ADAM10 and ADAM17, which constitutively cleave c-Met, inducing the release of soluble c-Met. ADAM10/17 pharmacological inhibition or gene silencing reduces c-Met shedding, but has limited impact on surface c-Met, which is overexpressed. Importantly, ADAM10 silencing inhibits UM cell invasion driven by FCS or HGF, while ADAM17 silencing has a limited effect. Altogether our data indicate that ADAM10 has a pro-invasive role and may contribute to UM progression.

Introduction Uveal melanoma (UM) is a rare ocular tumor that in up to 50% of cases develops into a metastatic disease, mostly in the liver (Coupland et al., 2013; Harbour et al., 2013; Martin et al., 2013; Singh et al., 2005). Metastases are usually identified 2 to 5 yr after treatment of the primary tumor and are poorly sensitive to chemotherapy, thus leading to a high mortality rate (Damato, 2010). The peculiar liver tropism of metastases and the identification of specific genetic markers of UM support the concept

that it is a distinct disease from cutaneous melanoma (Cree, 2000). Indeed, monosomy of chromosome 3 and amplification of chromosome 8q are aberrations that correlate with a poor prognosis in UM (Damato, 2010; Harbour, 2012). More recently, mutually exclusive mutations of the GNAQ and GNA11 genes (Van Raamsdonk et al., 2009, 2010), on one hand, and of the BAP1 (Wiesner et al., 2011), SF3B1 (Harbour et al., 2013; Martin et al., 2013), and EIF1AX (Martin et al., 2013) genes, on the other, have been identified as genetic markers of UM. Differently, mutations of Tp53,

Significance High expression of ADAM10 and, to a lesser extent, ADAM17 genes correlates with a short progression-free survival in uveal melanoma patients. In addition, pharmacological inhibition or gene silencing of ADAM10 inhibits in vitro invasion. These data indicate, for the first time, an involvement of ADAM10 in uveal melanoma progression and suggest that ADAM10 should be further studied as a potential target for therapy.

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BRAF, NRAS, CDKN2 (Triozzi et al., 2008), which are common in cutaneous melanoma, and mutations of TERT (Dono et al., 2014; Griewank et al., 2013) are very rare in UM. Studies of gene expression profiling of primary UM defined a gene signature associated with high risk of progression and identified two classes of patients with different prognosis (class-1 low risk and class-2 high risk) (Onken et al., 2006; Tschentscher et al., 2003). Moreover, several signaling molecules may play a role in metastatic progression of UM, among which the receptor for hepatocyte growth factor (HGF), c-Met deserves particular interest (Mallikarjuna et al., 2007; Peruzzi and Bottaro, 2006). In this respect, c-Met plays an important role in UM growth, migration, and metastatic potential, and may represent a potential target for therapy (Surriga et al., 2013; TopcuYilmaz et al., 2010; Wu et al., 2012). In addition, primary UM prone to progression overexpresses syntenin-1, a multiple docking protein involved in pro-invasive signals mediated by HGF through its receptor c-Met (Gangemi et al., 2012). A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are known to be upregulated in several types of malignancies and participate in progression (Kenny, 2007; Ko et al., 2007; Xu et al., 2010; Zhou et al., 2006). In particular, ADAM10 is highly expressed in cutaneous melanoma (Lee et al., 2010), where it plays a role in invasion and is considered a potential target for therapy (Saftig and Reiss, 2011). ADAM10 proteolytic activities mediate tumor progression through the cleavage of several substrates, such as the L1 cell adhesion molecule (L1-CAM), CD44, E- and N-cadherin, betacellulin, Her2/ErbB2, and Notch (Saftig and Reiss, 2011). Similarly, ADAM17 cleaves membrane-bound cytokines and growth factors (including TNF-a, amphyregulin, and TGF-a), surface receptors such as Notch, and adhesion molecules such as ICAM1, VCAM1, and ALCAM (Kenny, 2007; Rosso et al., 2007), thus increasing tumor cell survival, migration, and invasiveness. However, ADAM10 and ADAM17 have also been involved in the proteolytic cleavage of the HGF-receptor c-Met. This activity generates a soluble c-Met extracellular domain, which acts as a decoy receptor and limits HGF-mediated pro-invasive signals (Schelter et al., 2010). In view of the previously reported role of c-Met in UM and the general involvement of ADAM10/17 in c-Met cleavage, one may hypothesize that high ADAM10 and ADAM17 activity could reduce c-Met molecules on the cell membrane and limit c-Metmediated invasiveness. As no data on the role of ADAM10 in UM are reported, we here dissect the possible role of ADAM10, and of the closely related molecule ADAM17, in UM progression, HGF/c-Met-driven invasiveness, and regulation of membrane c-Met expression.

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Results High ADAM10, ADAM17, or MET gene expression in primary uveal melanoma correlates with metastatic risk To analyze the association between ADAM10 and ADAM17 gene expression with the development of metastases in human UM, we combined our microarray dataset (Amaro et al., 2013) and a similar dataset reported by Laurent et al. (2011), (total n = 108 UM cases) and normalized the data to avoid batch effects. The relation between ADAM10 or ADAM17 expression and survival was analyzed by COX regression, and Kaplan–Meier survival curves were plotted for cases above (‘high’) and below (‘low’) the median expression values (Figure 1). High expression of these genes correlates with poorer survival with a hazard ratio of 2.732 (P = 0.00041, 95% CI 1.528–4.88) for ADAM10 and 1.923 (P = 0.021 95% CI 1.09–3.38) for ADAM17. Three years after diagnosis, 44 of 54 patients (81%) with low ADAM10-expressing tumors were metastasis-free as opposed to 27 of 54 (50%) of high ADAM10-expressing cases (Figure 1A). These values dropped to 70% and 37% after 6 yr. This difference was less marked for ADAM17 expression (Figure 1B) with a 6-yr survival rate of 67% and 41% for low- and high-expressing tumors, respectively. The HGF-receptor c-Met is expressed in UM where it plays a role in invasiveness (Economou et al., 2005; Mallikarjuna et al., 2007). In agreement with these previous data, we found that expression of MET gene above the median value correlates with a short metastasis-free survival, with a hazard ratio of 2.6 (P = 0.0009, 95% CI 1.45–4.55) in the 108 primary UM (Figure 1C). ADAM10, ADAM17, and c-Met expression in uveal melanoma tumors and cell lines Immunohistochemistry of sections from 19 primary UM confirmed protein expression of ADAM10. Both tumor cells and histiocytes/macrophages showed expression of ADAM10, which appeared variable among different cases (Figures 2 and S1). Eleven of 19 samples were positive for ADAM10 expression, and eight were negative. ADAM10 expression was associated with a more rapid metastatic progression, relative to negative samples (Figure S2). These findings are suggestive of a role of ADAM10 in metastatic progression although the limited number of cases available for analysis did not allow achieving statistical significance. Immunohistochemical analysis of ADAM17 expression also showed staining of tumor cells and, in some samples, of histiocytes/macrophages (Figures 2 and S1). Overall, the expression of ADAM17 appeared weaker than that of ADAM10 as only in two cases, a clear positivity was found, while in most cases, expression was weak or undetectable. In agree-

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Figure 1. Kaplan–Meier analysis of ADAM10, ADAM17, and MET mRNA expression and cumulative probability of disease free survival (metastasis free survival) in patients with uveal melanoma. Patients (n = 108) with low expression (dark line, low) showed longer survival than patients with high expression (gray line, high). The cutoff is the median value. (A) ADAM10 (HR 2.732; 95% CI 1.528–4.88 P = 4.09E-04); (B) ADAM17 (HR 1.923; 95% CI 1.09–3.38 P = 0.021); (C) MET (HR 2.6; 95% CI 1.45–4.55 P = 0.0009) mRNA expression in uveal melanomas (n = 108).

ment with previous reports (Economou et al., 2005; Mallikarjuna et al., 2007), c-Met was expressed in primary UM, as 11 of 19 cases showed intense expression. Of note, c-Met expression was frequently observed in the same tumor areas displaying ADAM10 (Figures 2 and S1). In addition, the percentage of c-Met positive cells correlated with that of ADAM10-expressing cells 1140

(r = 0.586; P = 0.02), in 15 tumors that could be analyzed for both markers (Figure S3A). The use of a combined score (Gangemi et al., 2012), which considers staining intensity and percentage of positive cells, showed a trend toward a significant correlation (r = 0.50; P = 0.055) between c-Met and ADAM10 expression (Figure S3B). Furthermore, a striking correlation between ADAM10 and MET gene expression (r = 0.81; P = 0.0001) was found by analyzing the gene expression profiles of the 108 primary UM (Figure S3C). Next, we analyzed ADAM10 gene expression in four UM cell lines by Q-RT-PCR (Figure 3A). OMM1 and OMM2.5, derived from a cutaneous and a liver metastasis, respectively, displayed higher levels of ADAM10 mRNA compared to MEL270 and 92.1, obtained from primary tumors. In addition, Western blot analysis of the same cell lines demonstrated the presence of both the 102 kDa proenzyme and the 75 kDa active form of ADAM10 (Figure 3B). It is noticeable that all the UM cell lines strongly expressed the 75 kDa mature form of ADAM10, suggesting an elevated level of constitutive activation. As ADAM10 may be present at the cell membrane (Schelter et al., 2010), we stained live UM cells with an antibody directed against the extracellular domain of ADAM10 and analyzed its expression by flow cytometry. As shown in Figure 3C, all cell lines showed high expression of ADAM10 at the cell membrane. ADAM17 proenzyme (130 kDa) was also detected by Western blot in all cell lines (Figure S4), and three of four cell lines also expressed the mature form (80 kDa) of ADAM17. Western blot analysis confirmed expression of c-Met protein in whole cell lysates of the four UM cell lines (Figure 4A). In addition, we detected high levels of c-Met on the cell membrane of UM cell lines by immunofluorescence and FACS analysis of live cells (Figure 4B). ADAM10, ADAM17, and c-Met were then studied in a pseudo-metastatic model of UM obtained by transplantation of 92.1 cells into the spleen of NOG mice (Gangemi et al., 2012). Immunohistochemical analysis of human ADAM10 or c-Met showed moderate expression in spleen tumors, the primary site of implant, while liver metastases stained more intensely, and the normal hepatic tissue was negative (Figure 5). The analysis of consecutive liver sections showed that ADAM10, ADAM17, and c-Met were expressed in the same metastatic areas and that c-Met was present also at the membrane of UM cells (Figure 5 inset). Role of ADAM10, ADAM17, and c-Met ectodomain release in uveal melanoma To verify whether ADAM10 or ADAM17 is indeed enzymatically active in the UM cell lines, we studied the relationship of their activity with the proteolytic release of the ectodomain of c-Met, which is a substrate for both of these ADAMs (Foveau et al., 2009; Schelter et al., 2010). We first evaluated the presence of soluble c-Met in the ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Role of ADAM10 in uveal melanoma invasiveness

Figure 2. Immunohistochemical analysis of ADAM10, ADAM17, and c-Met in tissue sections of primary uveal melanomas. Primary uveal melanomas were stained for ADAM10, cMet, and ADAM17 expression (purple staining), (original magnification 6309). Five representative cases are shown. Bar indicates 100 micron. The inset shows primary uveal melanoma stained by secondary antibody in the absence of primary antibody (negative control).

conditioned medium of UM cell lines by ELISA. Indeed, all cell lines released high amounts (ranging from 20 to 140 ng/ml) of soluble c-Met (Figure 4C) in their culture supernatant after overnight culture in subconfluent conditions. In addition, we measured the serum level of soluble c-Met by ELISA in 10 healthy donors, 19 nonmetastatic, and 9 metastatic patients, and found elevated levels of soluble c-Met in the latter group (Figure 4D). These findings suggest that ADAM10 and/or ADAM17 are enzymatically active in vitro and also in vivo during UM progression. To verify whether ADAMs mediate the proteolytic cleavage of the extracellular domain of c-Met, 92.1, and MEL270, UM cells were incubated in the presence of increasing doses of Compound 18 (from 0 to 100 lM), a small molecule inhibitor of ADAM10 and ADAM17 activity (Nuti et al., 2010). As shown in Figure 6A, the release of c-Met decreased in a dose-dependent manner, suggesting that ADAM10 and/or ADAM17 activity contributed to c-Met shedding in UM cells. Cell viability was not affected by the overnight treatment with Compound 18 as ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

assessed by MTT assay (Figure S5A). We further assessed the relative role of ADAM10 and ADAM17 in c-Met cleavage by siRNA-mediated silencing experiments. Western blot of the 92.1 cell line treated with ADAM10- or ADAM17-targeting siRNA showed a 65 and 85% reduction of ADAM10 and ADAM17 protein expression, respectively, relative to the cells treated with scrambled siRNA (Figure 6B). Silencing of either ADAM10 or ADAM17 reduced the release of soluble c-Met in the conditioned media of UM cells by 33 and 56.6%, respectively (Figure 6C), indicating that both ADAMs mediate c-Met cleavage. Pharmacological inhibition of ADAM activity or silencing of ADAM10 inhibits cell invasiveness As c-Met plays an important role in HGF-mediated cell scattering and invasion, the question arose whether ADAM10 and ADAM17 limit cell invasion through c-Met cleavage or have a pro-invasive role themselves in UM. To address this point, we performed in vitro invasion tests using 92.1 UM cells in the presence of HGF at 100 ng/ml 1141

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Figure 3. Uveal melanoma cell lines express high levels of ADAM10: (A) Q-RTPCR analysis of ADAM10 mRNA in uveal melanoma cell lines. Expression values are normalized on the mean of GAPDH gene expression. (B) Western blot analysis of uveal melanoma cell lines shows the proADAM10 (102 kDa) and the mature ADAM10 (75 kDa) form. a-tubulin is shown as loading control. (C) Flow cytometric analysis of membrane ADAM10 in uveal melanoma cell lines. The dotted line is the negative control.

as chemo-attractant. Upon overnight incubation, Compound 18 significantly inhibited matrigel-coated filter invasion in a dose-dependent manner (Figure 7A). To further dissect the role of ADAM10 and ADAM17, we silenced 92.1 UM cells for either ADAM10 or ADAM17 by siRNA transfection, and tested them in an invasion assay. To this end, cells were serum-starved for 18 h and then FCS (10%) or HGF (100 ng/ml) were used as chemo-attractants. Silencing of ADAM10 significantly inhibited the invasion of the matrigel-coated membranes by 61  11.5% (P = 0.0008) in the presence of FCS and by 67  28.3% (P = 0.01) in the presence of HGF. By contrast, silencing of ADAM17 reduced invasion by 36.8  56.5% (P = 0.3) and 27  36.8% (P = 0.5), when FCS or HGF was used as chemo-attractants, respectively (Figure 7B). Neither ADAM10 nor ADAM17 silencing inhibited UM cell proliferation (Figure S5B). Although we used a pool of siRNA optimized to reduce possible off-target effects (Jackson and Linsley, 2010), to further rule out these effects, we transfected 92.1 UM cell line with a second 1142

pool of anti-ADAM10 siRNA duplexes, which target different sequences. Under these conditions, ADAM10 silencing similarly inhibited the invasion of 92.1 cells by 55  21% (P = 0.03) (Figure S6A). In addition, silencing of ADAM10 in another UM cell line (OMM2.5) also resulted in a significant inhibition of invasion (65  8.5%, P = 0.02, Figure S6B). The finding that ADAM10 silencing inhibits rather than increases UM cell invasiveness in response to HGF appeared paradoxical considering its role in c-Met shedding. Indeed, the increase in soluble c-Met theoretically would inhibit the pro-invasive effect of HGF. However, we observed that silencing of ADAM10 did not significantly affect the membrane expression of c-Met in UM cell lines, possibly due to its high expression levels (Figure 7C).

Discussion Previous reports indicated that c-Met plays an important role in promoting UM progression (Surriga et al., 2013; ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Role of ADAM10 in uveal melanoma invasiveness

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Figure 4. Expression of cellular and soluble c-Met in UM cell lines and in the serum of patients. (A) Western blot analysis of c-Met expression in four uveal melanoma cell lines. b-actin is shown as loading control. (B) Flow cytometric analysis of membrane c-Met in uveal melanoma cell lines. The dotted line is the negative control. (C) soluble c-Met (sc-Met) detected in conditioned medium of uveal melanoma cell lines by ELISA. (D) sc-Met detected in the serum of healthy donors, control (n = 10), non-metastatic, NM-UM (n = 19) and metastatic, M-UM (n = 9) uveal melanoma patients by ELISA. **P < 0.005. Mean and standard deviation are indicated.

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Figure 5. Immunohistochemical analysis of ADAM10, c-Met, and ADAM17 in a pseudo-metastatic model of uveal melanoma. Murine splenic uveal melanoma (left) and liver metastases (right) consecutive sections (Original Magnification 1009) were stained with anti-ADAM10, anti-c-Met, or anti-ADAM17 antibodies. Upper right insets show the negative control, bar indicates 100 micron. The lower inset in the c-Met panel shows a magnification 2009 of the indicated area.

Topcu-Yilmaz et al., 2010; Wu et al., 2012). Moreover, it has been shown that ADAM10-mediated cleavage of membrane c-Met limits pro-invasive signals (Schelter ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

et al., 2010). Therefore, we initially hypothesized that high ADAM10 or ADAM17 activity could cleave c-Met and decrease pro-invasive signals in UM. On the contrary, 1143

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Figure 6. Inhibition or silencing of ADAM10 reduces soluble c-Met. (A) Overnight incubation of 92.1 and MEL270 cell lines with 10 or 100 lM of Compound 18 inhibits shedding of c-Met detected by ELISA. (B) Western blot of 92.1 cell line treated with specific siRNA for ADAM10 and ADAM17 shows a substantially reduced expression of ADAM10 and ADAM17 proteins, respectively, compared to cells treated with non-targeting (scr) siRNA. a-tubulin is used as loading control. Last lane on the right was relocated within the same blot. (C) sc-Met detected by ELISA assay in the conditioned medium of 92.1 cell line is significantly reduced in cells treated with ADAM10- or ADAM17-specific siRNA compared with non-targeting (scr) siRNA. *P < 0.05, **P < 0.005.

here we report a pro-invasive role of ADAM10 in UM, on the bases of in vitro blocking experiments and retrospective correlations of ADAM10 expression in primary tumors with outcome. 1144

Figure 7. Inhibition or silencing of ADAM10 reduces uveal melanoma cells invasiveness. (A) Incubation of 92.1 cell line with 10 or 100 lM Compound 18 inhibits the ability of uveal melanoma cells to invade matrigel-coated filters. (B) Silencing of 92.1 cells with ADAM10-targeting siRNA significantly reduces invasion of matrigelcoated membranes independently of whether 10% FCS or 100 ng/ml HGF was used as chemoattractants. Silencing of ADAM17 slightly reduces invasion without reaching significance. Mean of three independent experiments is shown. Results are shown as percentage of control. (C) Flow cytometric analysis shows that ADAM10 silencing induces a significant reduction of membrane ADAM10 expression (arrow) left panel, but only a very modest upmodulation of c-Met (arrow), right panel. The dotted line is the negative control (NC). *P < 0.05, **P < 0.005, ***P < 0.0005.

In this report, we show that primary UM expresses ADAM10 and ADAM17 and that high ADAM10 and, to a lesser extent ADAM17, mRNA expression levels correlate with metastatic progression. Indeed, 6 yr after diagnosis, only 37% of patients with high ADAM10 were metastasis-free versus 70% of patients with a low ADAM10 expression. We further show that UM cell lines express the active form of ADAM10 and ADAM17, which ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Role of ADAM10 in uveal melanoma invasiveness

cleave c-Met and promote soluble c-Met release. Indeed, siRNA gene silencing showed that both ADAM10 and ADAM17 mediate c-Met shedding, in UM cell lines. Importantly, ADAM10 and/or ADAM17 may be active also in vivo, as suggested by the higher c-Met levels in the serum of metastatic UM patients than in non-metastatic ones. This finding may lead to set up a further study on a larger cohort of patients to investigate the use of soluble c-Met as biomarker of UM progression, similarly to recent findings in non-small-cell lung cancer (Fu et al., 2013). Several previous studies showed that c-Met and its ligand HGF are important mediators of cancer cell spreading and invasion (Stella et al., 2010; Trusolino et al., 2010). Particularly, c-Met plays an important role in human UM as it induces an interconverted cell phenotype (Hendrix et al., 1998) and mediates migration, invasiveness, and increased metastatic potential in UM cell lines (Economou et al., 2005; Ko et al., 2007; Wu et al., 2012). Moreover, c-Met protein expression was found in a fraction of primary UM samples by immunohistochemistry (Topcu-Yilmaz et al., 2010), and a correlation of high c-Met expression levels with increased mortality was reported (Mallikarjuna et al., 2007). In addition, the tyrosine kinase inhibitor crizotinib prevents metastases development through c-Met inhibition, in a xenograft model of UM, further supporting the role of c-Met in metastatic progression of this tumor (Surriga et al., 2013). In agreement with these data, we show that high MET gene expression strongly correlates with metastatic progression in a pooled dataset of 108 primary UM. Previous studies indicated that the engagement of c-Met with the antibody DN30 induces surface c-Met cleavage through ADAM10 activation, which results in the downregulation of c-Met expression and inhibition of HGF effects in different tumor cell lines (Schelter et al., 2010). In addition, the soluble c-Met ectodomain may bind HGF and limit its activity by acting as a molecular decoy (Michieli et al., 2004). Therefore, it has been proposed that ADAM10 activation may take part in the antitumor effects of therapeutic anti-c-Met antibodies (Schelter et al., 2010). Other reports suggested that not only ADAM10 but also ADAM17 cleaves c-Met (Foveau et al., 2009). Therefore, ADAM10 and/or ADAM17 activities may dampen UM responsiveness to HGF through the proteolytic cleavage of c-Met. Indeed, the present data, through small molecule inhibitors or siRNA targeting ADAM10 or ADAM17, confirm that both ADAM10 and ADAM17 contribute to the proteolytic cleavage of c-Met in UM cell cultures and that blocking these enzymes resulted in a reduction of soluble c-Met production. However, inhibition of ADAM10 did not significantly modify the membrane expression of c-Met in cell lines, possibly due to c-Met overexpression. This finding was also supported by the expression of ADAM10/ADAM17 and cell membrane c-Met in the same areas of UM experimental metastases to the liver. In addition, human ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

primary UM frequently displays coexpression of ADAM10 and c-Met in the same tumors, although c-Met expression on the tumor cell membrane was difficult to assess. Moreover, inactivation of ADAM10 by siRNA silencing did not increase cell invasion in vitro in response to the c-Met ligand HGF. On the contrary, we show that silencing of ADAM10 significantly inhibited the invasiveness of UM cells triggered by FCS or even by HGF, while in these tests, ADAM17 silencing had limited effects. These data indicate a pro-invasive role of ADAM10 in UM cell lines in vitro. A pro-invasive role of ADAM10 was previously demonstrated in other cancers and was related to the enzymatic activity of ADAM10 (Saftig and Reiss, 2011). In addition, the correlation between ADAM10 expression and c-Met, at mRNA and protein level, suggests that both molecules contribute to a more malignant phenotype of UM. Altogether the high expression of ADAM10 in UM prone to metastasis and its pro-invasive role in vitro support a role of ADAM10 in UM progression. Therefore, ADAM10 targeting may deserve further preclinical investigation in UM models, possibly in combination with other agents. As the current chemotherapy regimens with dacarbazine or fotemustine show low response rates, new therapeutic options are needed. Indeed, several novel biological therapies, including inhibitors of protein kinase C, MEK, or AKT, or immune check-point blockers such as ipilimumab are currently being tested in metastatic UM (Buder et al., 2013). Finally, clinical trials using LY2801653, an orally bioavailable multikinase inhibitor with potent activity against c-Met (NCT01285037) or a humanized monoclonal antibody against c-Met, LY2875358 (NCT01287546), are recruiting patients.

Methods Cell cultures and reagents The human UM cell lines Mel 270, 92.1, OMM1, and OMM2.5 (De Waard-Siebinga et al., 1995; Luyten et al., 1996) were cultured in RPMI 1640 (Gibco Life Technologies, Waltham, MA, USA) supplemented with 10% fetal bovine serum. ADAMs inhibitor Compound 18 has been previously described (Nuti et al., 2010).

Patients and tissue samples Tissue and blood samples were obtained from UM patients after enucleation surgery with approval of the institutional board and informed written consent of the patients in accordance with the Declaration of Helsinki. We analyzed two previously described datasets of microarray gene expression (Amaro et al., 2013; Laurent et al., 2011) from the Gene Expression Omnibus (GEO) (www.ncbi. nlm.nih.gov/pubmed/). Patient and tumor characteristics of our dataset (Amaro et al., 2013) are described in Table S1, and information on the second dataset, GSE22138, is available in GEO. The CEL files (Affymetrix HGU133plus2 microarrays) of two datasets for a total of 108 samples were joined and normalized using the RMA procedure (Irizarry et al., 2003). For ADAM10 and ADAM17 expression, probe sets 202604_x_at and 205745_x s were used (see Table S2). Serum was collected from 19 non-metastatic (NM-UM), from 9 metastatic (M-UM) UM patients during follow-up (from 3 months to 3 yr post-primary tumor treatment) and from 10 healthy donors.

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Mouse model of UM

Migration and invasion assay

The experiments were performed according to the National Regulations and approved by the Institutional Review Board for Animal Experimentation and the Italian Ministry of Health. All mice were anesthetized with intraperitoneal injections of ketamine and xylazine. The luciferase gene-transduced UM cells 92.1 or Mel270 were implanted under the spleen capsule of NU/NU mice (Janvier, France) or NOD/SCIDIL2Rc null mice (Jackson Laboratory, Bar Harbor, ME, USA) as described (Gangemi et al., 2012). Mice were inspected by the IVIS (IVIS imaging 100; Xenogen, Caliper LifeSciences, Hopkinton, MA, USA) for tumor growth and underwent necropsy when liver metastases were detected.

BD BioCoat migration or invasion chambers coated with growth factor-reduced matrigel (BD Biosciences Italy, Milan, Italy) were used for invasion assays as described (Gangemi et al., 2012). Cells were cultured in medium containing 1% FCS for 18 h before the assay. Recombinant HGF (100 ng/ml; Peprotech Tebu-Bio Italy, Milan, Italy) or 10% FCS was added to the bottom chambers. After 36-h incubation, migrated cells were fixed and stained with 1% toluidine blue to visualize nuclei. The numbers of migrating cells in a minimum of 30 fields were counted, and the mean for each chamber determined. Experiments were run in triplicate.

Immunohistochemistry Immunohistochemical detection of ADAM10 (ab1997; Abcam, Cambridge, UK), ADAM17 (ab39163; Abcam), or c-Met (MET c-12; Santa Cruz Biotechnology, Dallas, TX, USA) was performed on formalinfixed, paraffin-embedded tissue sections of 19 primary UM as previously described (Gangemi et al., 2012). Clinical and pathological characteristics of 14 patients studied are described in Table S1 while 5 additional patients not included in the microarray study are described in Table S3. For murine sections, the EnVision+SystemHRP and DAB+ (Dako) were used.

RT-QPCR Quantitative RT-PCR analysis was performed as described (Gangemi et al., 2012). The ADAM10 upper primer was GAGGAGTGTAC GGTGTGCCAGTT, and lower primer was GACCACTGAAGTGCC TACTCCA. Relative quantification of mRNAs was calculated by the DCt method.

FACS analysis Cells were incubated with anti-ADAM10 (R&D) or c-Met (Cell Signaling) antibodies, followed by DyLight488 Goat anti-rabbit or anti-mouse secondary antibody. Cell fluorescence was analyzed in a FACScan (Becton & Dickinson Italy, Milan, Italy).

Acknowledgements We wish to thank Dr Cecile Laurent for providing information on microarray data; Dr. Alessandro Poggi, Dr. Maria Raffaella Zocchi, and
for providing reagents; Mr. Francesco Campelli for Dr. Roberta Vene technical help; and Fondazione Compagnia di San Paolo, AIRC (Associazione Italiana per la Ricerca sul Cancro) IG 13518. AA is recipient of a fellowship PO CRO Fondo Sociale Europeo Regione Liguria 2007-2013 Asse IV “Capitale Umano”.

References Western blot analysis Western blot of cell lysates was performed as previously described (Gangemi et al., 2012). c-Met was detected by mouse anti-c-Met monoclonal antibody (Cell Signaling, Danvers, MA, USA), while ADAM10 and ADAM17 were detected by rabbit anti-ADAM10 and ADAM17 antibodies (Abcam). Anti-b-actin and anti-a-tubulin monoclonal antibodies (Sigma-Aldrich, Milan, Italy) were also used.

ELISA for C-Met Cell supernatants and patients sera were tested with commercially available ELISA kit for human c-Met (DuoSet; R & D Systems, Minneapolis, MN, USA).

Statistical analysis Disease-free survival curves were constructed using the Kaplan– Meier method and compared with the Wilcoxon log-rank test. The one-way ANOVA and appropriate multiple comparison tests were used to compare expression levels between patients and control subjects. The paired Student’s t-test was used when appropriate. A P-value lower than 0.05 was considered significant. Analyses were performed using PRISM 5 (Graph-Pad Software, San Diego, CA, USA).

Small interfering RNA (siRNA) transfection ON-TARGET plus SMART pool for human ADAM10, ADAM17, or siCONTROL non-targeting siRNA pool (Dharmacon, Lafayette, CO, USA) were transfected in 92.1, Mel270, and OMM2.5 cells kept in serum-free Opti-mem medium (Gibco, Life Technologies) using Lipofectamine 2000 Reagent (Invitrogen Life Technologies) following the manufacturer’s instructions. In a second set of experiments, the IBONI siRNA-Pool (RIBOXX Life Sciences, Radebeul, Germany)) targeting different coding sequences of ADAM10 was also used.

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Supporting information Additional Supporting Information may be found in the online version of this article: Figure S1. Immunohistochemical analysis of ADAM10, ADAM17 and c-Met in additional tissue sections of primary UM.

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Figure S2. Kaplan–Meier analysis of ADAM10 protein expression and metastasis-free survival in patients with primary tumors. Figure S3. Analysis of the correlation between ADAM10 and c-Met protein and mRNA expression. Figure S4. Western blot of ADAM17 in UM cell lines. Figure S5. Proliferation of UM cells treated with compound 18 or with siRNA anti-ADAM10.

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Figure S6. Effect of siRNA silencing on protein expression. Table S1. Patients and tumor characteristics. Table S2. Description of affymetrix probesets for ADAM10 and ADAM17 genes. Table S3. Description of 5 patients whose primary tumors were used in immunohistochemistry analysis but were not included in the microarray.

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