Novel Alternatively Spliced ADAM8 Isoforms Contribute to the Aggressive Bone Metastatic Phenotype of Lung Cancer

Oncogene (2010) 29, 3758–3769 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Novel alternatively spliced ADAM8 isoforms contribute to the aggressive bone metastatic phenotype of lung cancer

I Herna´ndez1, JL Moreno2, C Zandueta1, L Montuenga3,4 and F Lecanda1

1Adhesion and Metastasis Laboratory, Division of Oncology, University of Navarra, Pamplona, Spain; 2Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, USA; 3Department of Histology and Pathology, School of Medicine, University of Navarra, Pamplona, Spain and 4Biomarkers Laboratory, Center for Applied Biomedical Research (CIMA), University of Navarra, Pamplona, Spain

ADAMs (a disintegrin and metalloprotease) are trans- survival rates for lung cancer are o15% in all developed membrane proteins involved in a variety of physiological countries. It is estimated that 30–40% of lung cancer processes and tumorigenesis. Recently, ADAM8 has been patients with advanced NSCLC suffer from bone associated with poor prognosis of lung cancer. However, metastasis (Coleman, 1997). Patients with bone metas- its contribution to tumorigenesis in the context of lung tasis experience pain, metabolic syndromes and spinal cancer metastasis remains unknown. Native ADAM8 cord compression associated with pathological fractures expression levels were lower in lung cancer cell lines. as a consequence of osteolytic lesions. In contrast, we identified and characterized two novel ADAMs (a disintegrin and metalloprotease) form a spliced isoforms encoding truncated proteins, D18a and large family of cell-surface proteins, which are char- D140, which were present in several tumor cell lines and not acterized by disintegrin and metalloproteinase domains, in normal cells. Overexpression of D18a protein resulted in that possess adhesive properties and proteolytic activ- enhanced invasive activity in vitro.ADAM8anditsD140 ities, respectively (Lu et al., 2007). The majority of isoform expression levels were markedly increased in lung ADAMs require removal of their prodomain by cancer cells, in conditions mimicking tumor microenvironment. proprotein convertases to become catalytically active. Moreover, addition of supernatants from D140-overexpressing Other typical domains include the cysteine-rich, epider- cells resulted in a significant increase in tartrate-resistant acid mal growth factor -like, transmembrane and short phosphatase þ cells in osteoclast cultures in vitro.These cytoplasmic domains (Seals and Courtneidge, 2003). findings were associated with increased pro-osteoclastogenic Over 40 different ADAMs have been identified that cytokines interleukin (IL)-8 and IL-6 protein levels. Further- have important roles in extracellular matrix remodeling, more, lung cancer cells overexpressing D140 increased cell migration and signaling. ADAMs modulate extra- prometastatic activity with a high tumor burden and increased cellular signals through their sheddase activity by osteolysis in a murine model of bone metastasis. Thus, the processing several membrane-bound proteins including expression of truncated forms of ADAM8 by the lung cancer cytokines, growth factors and their receptors (Seals and cells may result in the specific upregulation of their invasive Courtneidge, 2003). In the context of lung cancer, the and osteoclastogenic activities in the bone microenvironment. metalloprotease (MMP) domains of ADAM10 and These findings suggest a novel mechanism of tumor-induced ADAM17 are key components in the epithelial-derived osteolysis in metastatic bone colonization. growth factor receptor signaling cascade that mediate Oncogene (2010) 29, 3758–3769; doi:10.1038/onc.2010.130; proteolytic shedding of its membrane-tethered ligands published online 10 May 2010 (Blobel, 2005, Sahin et al., 2004). Recent studies have focused on ADAM8, which is Keywords: microenvironment; tumor–stroma; splicing; expressed in the immune and central nervous systems osteolysis; colonization (Hooper and Lendeckel, 2005). It contains a proline-rich SH3 cytoplasmic tail suggesting that it interacts with other cytoplasmic proteins (Yoshida et al., 1990). In contrast to several other related molecules, ADAM8 Introduction is activated by its autocatalytic activity (Schlomann et al., 2002). Conditional deletion of ADAM8 shows no Lung cancer is the leading cause of cancer death apparent phenotype, despite its marked expression in throughout the world (Jemal et al., 2002). The 5-year bronchioepithelial cells in the lungs, salivary glands and kidney (Kelly et al., 2005). ADAM8 has been shown to Correspondence: Dr F Lecanda, Adhesion and Metastasis Laboratory, participate in the allergic inflammatory response (Foley Division of Oncology, Center for Applied Biomedical Research et al., 2007, King et al., 2004, Matsuno et al., 2006). (CIMA), Pio XII-55, University of Navarra, Pamplona, Navarra in vitro ADAM8 has been shown to process CD23 31080, Spain. E-mail: fl[email protected] (Fourie et al., 2003), myelin basic protein (Amour et al., Received 23 September 2009; revised 9 March 2010; accepted 25 March 2002) and a peptide derived from interleukin (IL)-1 type 2010; published online 10 May 2010 II receptor (Naus et al., 2006). Mechanism of bone colonization I Hernańdez et al 3759 ADAM8 is upregulated in various tumors including of 95 amino acids. The new open reading frame stopped lung and brain tumors (Wildeboer et al., 2006), and is at a TGA codon within the exon 19. The sequence considered to be a prognostic marker for renal cell comparison between wild-type ADAM8 and its D18a carcinomas and the best predictor of distant metastasis isoform revealed that D18a contained an additional (Roemer et al., 2004a b). Recently, it has been suggested exon 18 of 179 bp. This additional exon (exon 18a) was that the expression of ADAM8 may be an early prognostic flanked by canonical exon acceptor and donor sites marker for lung adenocarcinomas (Ishikawa et al., 2004). (Figure 2b). The divergent carboxy terminal of D18a It has also been shown that ADAM8 stimulates osteo- showed no similarity to any other proteins in the clast differentiation (Choi et al., 2001). Thus, we sought common databases. In all, five potential N-linked to investigate the involvement of lung tumor-derived glycosylation sites (NX; S/T) were present, four of ADAM8 in the mechanisms of osteolysis and bone which were also found at the same position in native destruction during lung cancer metastasis to bone. ADAM8, whereas the fifth was located at position 698 In this study, we have identified several differentially within the new carboxy terminal (Figure 2b). truncated ADAM8 isoforms in human lung tumor cells The other splice variant D140, contained an open that confer different invasive properties in vitro. Differ- reading frame of 1620 nucleotides encoding 539 amino ential regulation of these isoforms was observed under acids (B59 kDa). The D140 isoforms encompassed 14 the conditions that mimicked tumor–stroma interac- exons and retained intron 14. The retention of intron 14 tions. The D140 isoform markedly increased the osteo- introduced a novel frameshift with a premature TAA clastogenic activity in an in vitro assay. Furthermore, stop codon. The last 56 nucleotides of the D140 open D140-overexpressing cells induced bone metastasis with reading frame encoded a novel 17 amino-acid sequence increased tumor burden and tumor-induced bone with no similarity to any other known protein (Figure 2c). osteolysis in an in vivo model of metastasis. These In all, three out of four N-linked glycosylation sites were findings suggest that ADAM8 alternative splicing similar to the wild-type ADAM8 (Figure 2c). variants participate and contribute to the deleterious effects of tumor-derived bone metastasis. Expression of hADAM8 isoforms in cell lysates and culture supernatants We sought to compare the protein levels of ADAM8 in tumor cell lines and normal NHBE cells. Owing to the Results complexity of ADAM8 protein processing, immuno- blotting analysis was carried out with different anti- Characterization of ADAMs in lung cancer cell lines bodies on cell lysates and culture supernatants. The In a long-term project searching for alternative splice most reliable results were obtained with an antibody variants of ADAM proteins altered in lung cancer, we recognizing the MMP domain. Several immunoreactive focused in ADAM8, as it has been found elevated in bands were consistently detected for ADAM8: a B90 lung cancer patients (Ishikawa et al., 2004). We carried kDa ‘processed form’ that was catalytically active, out a conventional semiquantitative reverse transcrip- consistent with propeptide removal; and a ‘remnant’ tase–PCR strategy in a battery of human lung tumor cell form with an apparent Mr of B60 kDa (Schlomann lines and primary normal human bronchial epithelial et al., 2002). All these bands represented membrane- (NHBE) cells. Using primers for ADAM8, we found bound forms of ADAM8 (Figure 3a). The processed that two isoforms were amplified. The bottom band form was not observed in SCLC cell lines, whereas it corresponded to a coamplification of two species: the was detected in NHBE and in all NSCLC cell lines expected native ADAM8 amplicon and a novel splicing tested. In contrast, the remnant form, absent in NHBE variant, which we called DADAM8–18a (D18a) cells, was detected in all SCLC and NSCLC cell lines (Figure 1a). The other amplified transcript (top band) tested (Figure 3b). represented another novel alternatively spliced isoform, In the conditioned medium, only a B65 kDa isoform named DADAM8–140 (D140). Full-length DADAM8– that has been previously described (Mandelin et al., 18a (D18a) and DADAM8–140 (D140) were cloned and 2003) (recognized by the A8h-MD antibody) was sequenced. detected in the nontransformed epithelial lung cell line To more accurately assess the relative levels of the (NHBE) (Figure 3c). new isoforms, we used quantitative real-time PCR with specific primers to each isoform. We found that D140 isoform was upregulated in small cell lung cancer Overexpression and inhibition of ADAM8 isoforms (SCLC) cell lines as compared with primary NHBE in A549 and H460 cells cells. In contrast, D18a was more abundantly expressed To examine the roles of different isoforms of ADAM8, in some adenocarcinoma cell lines (Figures 1b). The we retrovirally transduced A549 and H460 cell lines with exon–intron organization of ADAM8 is shown in constructs encoding native ADAM8 (wild-type A8) and Figure 2. The full-length D18a complementary DNA its DADAM8–18a (D18a) and DADAM8–140 (D140) containing an open reading frame spanning 2238 isoforms. Higher levels of 75-kDa protein were detected nucleotides encoded 745 amino acids (B80 kDa). From in wild-type A8-transfected cells as compared with the point of divergence with the native ADAM8, 290 mock-transduced cells using western blot analysis with nucleotides encoded the new carboxy terminal domain the MD (metalloprotease domain) antibody (Figure 4a).

Oncogene Mechanism of bone colonization I Herna´ndez et al 3760 SCLC NSCLC

M NHBE H69 H82 H209 H510 N417 H23 HCC44 SKLU1 PC14 HCC827 H358 A549 H322 HCC15 CALU-1 LUDLU-1 H1703 H157 SK-MES1 H1299 H460 H661 H720 H727

ADAM8

β-actin

Δ14´ 5

1 Relative fold change

Relative fold change 1 0

H69 H82 H23 H209H510 N417 H358A549 H322 H157 H460 H661 H720H727 NHBE PC14 H1703 H1299 HCC44SKLU1 HCC15CALU-1 HCC827 LUDLU-1 SK-MES-1 SCLCAdenocarcinoma Squamous Large Carcinoid Cell Carcinoma Cell Figure 1 Expression profiles of several ADAMs and ADAM8 spliced isoforms. (a) Expression levels of ADAM8 were assessed by semiquantitative reverse transcriptase–PCR on a battery of tumor human cell lines and NHBE. The top band corresponds to D140 isoform, whereas bottom band correspond to a coamplification of D18a and native ADAM8. (b) Quantification of expression levels for two ADAM8 spliced variants, D18a and D140 were assessed by real-time quantitative PCR.

In D18a-transduced cells a B70-kDa isoform was D140 isoform, it appears consistent that the upregulation detected together with overexpression of the 75-kDa of D140 isoform results as a compensatory mechanism in isoform. In both H460 and A549 cells transduced with response to decreased native ADAM8. D140,aB68-kDa band was detected with a slight increase in the expected molecular weight presumably due to glycosylations. No changes were observed in the The invasive activity of the ADAM8 isoforms conditioned media of transduced cells (data not shown). We studied the invasive properties of different ADAM8 For knockdown experiments, we stably transfected isoforms in vitro in two different cell lines H460 and A549 A549 and H460 cells with the ADAM8 vector contain- in a Boyden chamber assay. As a chemoattractant, we ing two different small interfering RNA (siRNA) used conditioned medium derived form murine ST-2 constructs or a scrambled nonspecific siRNA sequence. stromal cells. A marked increase in the invasive activity of Western blot analysis revealed robust inhibition of the H460 and A549 cells was detected in cells, overexpressing 75 kDa processed isoform in cell lysates (Figure 4b). native ADAM8 and its D18a isoform (Po0.001), as Densitometric analysis of the reactive bands revealed a compared with the mock-transduced and parental cells decrease between 80 and 90% for A549 cells, and 70 and (Figure 5a). In contrast, the invasive ability of D140- 80% for H460 cells. Interestingly, inhibition of ADAM8 transduced cells was significant (Po0.05), whereas in in both cell lines led to a concomitant increase in the A549, D140-transduced cells showed a dramatic increased D140 isoform, which was stronger in A549 cells as invasive activity (Po0.0001). Knockdown of ADAM8 compared with H460 cell line, whereas D18a expression using two different siRNA in both cell lines showed a levels in knockdown transfectants were cell-line depen- significant decrease in invasiveness as compared with dent. (Figure 4c). Although both siRNA targets (1 in control cells (Figure 5b). These results suggest that exon 17 and the other in exon 19) could theoretically ADAM8, and both D18a and D140 isoforms contribute inhibit native ADAM8 and D18a isoforms, but not the to increase invasiveness in lung cancer cells.

Oncogene Mechanism of bone colonization I Herna´ndez et al 3761 SP PD MD DD CRD ED TD CTD

hADAM-8

5’ 123456 7 8 9 10 11 12 13 14 15 16171819 2021 2223 3’

Δ 14’

Δ 18a

Δ 18a Δ 14’

5’ 132 41251415176 7 8 9 10 11 13 16 1818a 19 20 21 22 23 3’ 5’ 1 2354126 7 8 9 10 11 13 14-bis 3’

GTGTGCAACCACAAGCAGGAGTGCCACTGCCACGCGGGCTGGGCCCCGCCCCACTGCGCGAAGCTGCTGACTGAGGTGCA GTGAAGCCGGCTGGTGAGCTGTGCCGTCCCAAGAAGGACATGTGTGACCTC CGCAGgtaggcctccctggccagcgcaggtggggctggggtccaagctcagagggcccgaacagagcccaggcagaagccgcgggcccgggaagctggtggtgttcacggtg GAGGAGTTCTGTGACGGCCGGCACCCTGAGTGCCCGGAAGACGCCTTCCA ccagtttgctgccacaggcatcgacacacaggtataggcagagcctgtctgtccccaggctgatgtgaggaggggagagccctgtcccagagggaggcagtggccgagggcaggg GGAGAACGGCACGCCCTGCTCCGGGGGCTACTGCTACAACGGGGCCTGTC cacggccgtgagctgagactccagtgtcctcagcctgtgtcttccagCCCCTCCTCGGCCCGGGGCCCTGAGGCCACCCAGCATTTCCTCCCCGACG CCACACTGGCCCAGCAGTGCCAGGCCTTCTGGGGGCCAGGTGAAGTGGGC GCCACTGAGCATGTGGTCGTCCAGAGCATCAGGCCTCAAAGGCACAGCCGTGCCCACAGCACTGTGGCCCAGGGCCACCCT ACAGAGCTGGCCTGGCAGGGTGGCTGCAGGCCTTGCCAGGGCTAACCCGC GCCAAGCTCTCCAACCTGTCACTGGCAAGCTGGGAGTGCCAGTGTAAGgtaccaggtcctccctgaggggtccacagggcacagcctgggggtgct TCCTGGTCCCACAGGTGGGCAGGCTGCCGAGGAGTCCTGCTTCTCCTATGA gagaaagaccgtgtctctgggaacctccctggctcctgcctgaggtcgcggcctgcctgcctgatgtggggccgtgtgtcccacagCGTCCGGGAGCCTCCCCGTCCTC CATCCTACCAGGCTGCAAGGCCAGCCGGTACAG GTGGTGGTGGTTCTGGTGCTCCTGGCAGTTGTGCTGGTCACCCTGGCAGGCATCATCGTCTACCGCAAAGCCCGGAGCCGC ATCCTGA

SP PD MD DD CRDED ? SP PD MD DD CRD*

AKLLTEVHAAPPRPGALRPPSISSPTATEHVVVQSIRPQRHSRAHSTVAQ PCSGGYCYNGACPTLAQQCQAFWGPGEVGTE GHPAKLSNLSLASWECQCKRPGASPSSWWWFWCSWQLCWSPWQASS LAWQGGCRPCQG STAKPGAAS Figure 2 Cloning and sequencing of novel spliced isoforms of ADAM8. (a) Schematic diagram of the genomic locus and the different domains of native ADAM8. Colored boxes represent different protein domains of ADAM8 and black boxes represent the 50 and 30 untranslated regions (UTRs). Black arrows (-) represent the primers used in the initial screening detecting native ADAM8, D18a and D140. Small line (À) represents sequences used for siRNA interference. (b)ADAM8D18a exon–intron structure with the new intron resulting from alternative splicing depicted in red (exon 18a). The sequences of exons 18 and 19 (green), 18a (red) and the intronic sequence (lower black case) are shown. The acceptor and donor splice sites are underlined and the stop codon is shown. The amino acids of a new N-glycosylation site are underlined. (c)ADAM8D140 exon-intron structure with the new intron resulting from alternative splicingdepictedinaredbox(the new exon 14-bis). The sequence of the exon 14-bis is shown (red) between exons 14 and 15 (green) and the stop codon (black). The different domains of wild-type ADAM8 are shown as different colors: signal peptide (SP), prodomain (PD), metalloprotease domain (MD), disintegrin domain (DD), cysteine-rich domain (CRD), EGF-like domain (ED), transmembrane domain (TD), cytoplasmic tail domain (CTD). The potential N-glycosylation sites are indicated with a mark ( ). CRD* means the novel aminoacid sequence in this domain.

SCLC NSCLC NHBE1 2 3 4 5 6 NHBE1 2 3 4 5 6 7 SP PDMD DD CRD EGF TMCT 75 ADAM8 A8-MD

Extracellular Intracellular 50

PROCESSED FORM ∼90 kD 37 β-actin REMANENT FORM ∼50 kD SCLC NSCLC SOLUBLE FORM ∼50 kD NHBE1 213 4 5 6 NHBE2 3 4 5 6 7 68 A8-MD 42 Figure 3 Western blot analysis of the ADAM8 isoforms. (a) Schematic of different ADAM8 isoforms that have been previously characterized. Symbols correspond to the isoforms detected by Western blot analysis. (b) Western blot analysis of ADAM8 in lysates of normal lung epithelial and lung tumor cell lines detected with the A8h-MD antibody directed to the metalloprotease domain (MD) epitope. (c) Western blot analysis of ADAM8 in conditioned media. SCLC cell lines: 1 (H69), 2 (H187), 3 (H345), 4 (H510), 5 (H82), 6 (H209). NSCLC cell lines: 1 (H23), 2 (A549), 3 (H2087), 4 (H157), 5 (H460), 6 (H661), 7 (H727).

Regulation of ADAM8 and its isoforms by tumor–stroma regulation of ADAM8, human H460 and A549 cells interactions were cocultured with murine stromal ST-2 cells to mimic Tumor–stroma interactions have been shown to be the tumor–stroma microenvironment. Under these critical for tumor initiation, progression and metastatic conditions, D140 expression levels were markedly development (Hanahan and Weinberg, 2000). Thus, to upregulated in H460 cells and slightly increased in determine the role of cell–cell interactions in the A549 cells, whereas D18 levels remained unchanged

Oncogene Mechanism of bone colonization I Herna´ndez et al 3762 scramble siRNA1 siRNA2 Vector Native A8 Vector Δ18 Vector Δ14´ scramble siRNA1 siRNA2 A549 Δ14´ 75 75 75 75 1 1.46 1.96 A8-MD Δ 50 18a 50 50 50 1 0.2 0.1 1 1.37 1.15 β-actin β-actin 37 37 37 37

scramble siRNA1 siRNA2 Δ Vector Δ14´ scramble siRNA1 siRNA2 H460 Vector Native A8 Vector 18 Δ14´ 75 75 75 75 1 1.1 1.4 A8-MD Δ18a 50 50 50 1 0.2 0.3 1 0.62 0.7 50 β-actin β-actin 37 37 37 37 Figure 4 Overexpression and knockdown of ADAM8 isoforms. (a) Wild-type ADAM8 and its D18a and D140 isoforms were cloned into pBabe-Neo and transduced into H460 and A549 lung tumor cell lines. ADAM8 isoforms were detected by western blot analysis using the A8h-MD antibody. Full-length ADAM8 (%), D18a (v) and D140($) are shown in lysates. (b) Several targets against ADAM8 were designed as siRNA and transfected into H460 and A549 cells. A nonspeciﬁc siRNA sequence (scramble) was used as the negative control. ADAM8 expression was analyzed by western blot analysis using the A8h-MD antibody. (c) Expression levels of D18a and D140 after knockdown of full-length ADAM8 were assessed by semiquantitative reverse transcriptase–PCR. Numbers denote the densitometric analysis carried out using AnalySIS image analysis system.

250 *** A549 A549 A549 *** 60 200 ST-2 +-+ *** 150 40 * Δ14´ 100 ** 20 1 1.44 50 % Invaded Area % Invaded Area β-actin 0 0 Control A8 Δ18a Δ14´ Scramble siRNA1 siRNA2 H460 80 400 H460 *** H460 ST-2+- + 60 ** Δ 300 * * 14´ * 40 200 0 1 β 100 20 -actin % Invaded Area % Invaded Area 0 0 Control A8 Δ18a Δ14´ ScramblesiRNA1 siRNA2 Figure 5 Invasion and coculture experiments. (a) Invasion assays using H460 and A549 cells overexpressing wild-type ADAM8, D18a and D140 isoforms, and wild-type ADAM8 knockdown transfected cells were carried out in a Boyden chamber stimulated with conditioned medium of ST-2 cells. In both cell lines, overexpression of full-length ADAM8 and D18a increased invasion dramatically, whereas such effect was milder in D140 transduced cells. (b) Invasion was decreased in ADAM8 knockdown cells in H460 and A549 cell lines under these experimental conditions. (c) H460 and A549 cells were incubated with medium alone or in the presence of the murine marrow stromal cell line ST-2 for 3 days after confluency. Specific semiquantitative reverse transcriptase–PCR was used to assess human gene expression of the different isoforms. The D140 isoform was upregulated by cell–cell interactions in both cell lines. Experiments were repeated three times with identical results. Numbers denote densitometric quantification of the bands (*Po0.05, **Po0.01,***Po0.001).

(data not shown). These findings suggest that the effects receptor-activated nuclear factor-k B-ligand, the condi- of D140 might be exacerbated in conditions that promote tioned media from D140-expressing cells led to a tumor–stroma interactions (Figure 5c). significant increase in osteoclast differentiation for both H460 (Po0.001) and A549 (Po0.01) cells (Figure 6a). The conditioned media from native ADAM8 and its Effects of ADAM8 and its isoforms on osteoclastogenesis D18a isoform were not significantly different from the A soluble isoform of murine ADAM8 has been media of mock-transduced cells. previously implicated in osteoclastogenesis (Choi et al., Although native ADAM8 was expressed in low levels 2001). To examine the role of ADAM8 isoforms in in several lung cancer cell lines, whereas several splicing osteoclastogenesis, conditioned media from cells over- isoforms were upregulated in tumor cells as compared expressing native ADAM8, D18a or D140 isoforms, were with normal NHBE cells, we sought to determine the added to mouse bone marrow macrophages to study consequences of ADAM8 inhibition. Interestingly, their ability to induce osteoclast formation. In the conditioned media from native ADAM8 siRNA-inhib- presence of macrophage colon-stimulating factor and ited A549 and H460 cells led to a slight increase in the

Oncogene Mechanism of bone colonization I Herna´ndez et al 3763 A549 H460

Δ IL-8 14 ** Δ14 *** Mock Δ18a Δ18a

Native A8 Native A8 Δ14' Control Control *

0204060 0 5 10 15 0 200 400 600 800 2 6 Total TRAP+ Area (mm2) Total TRAP+ Area (mm ) fg/mL*10 cells

A549 H460 IL-8 Scramble siRNA1 * siRNA1 *

siRNA1 siRNA2 * siRNA2 *

scramble scramble 0 50000 100000 150000 fg/mL*106 cells 01020 30 40 0 5 10 15 Total TRAP+ Area (mm2) Total TRAP+ Area (mm2)

Control Native A8 Δ18a Δ14

Scramble siRNA1 siRNA2

Figure 6 In vitro osteoclastogenic assay. (a) Conditioned media from H460 and A549 mock- and D140-transduced cells were collected and incubated with murine bone marrow macrophages for 6 days in media containing macrophage colon-stimulating factor (20 ng/ml) and receptor-activated nuclear factor-k B-ligand (40 ng/ml). The conditioned media from D140-overexpressing cells induced the highest numbers of TRAP þ multinucleated cells. The conditioned media of knockdown ADAM8 cells induced a signiﬁcant increase in the number of multinucleated TRAP þ cells (Po0.05). The experiment was repeated three times with identical results. (b) Representative images ( Â 20) of TRAP þ cells of each condition induced by A549 conditioned media are shown. (c) IL-8 levels were measured by enzyme-linked immunosorbent assay in the conditioned media from D140-overexpressing A549 cells and ADAM8 siRNA-treated A549 cells. (*Po0.05, **Po0.01). number of multinucleated tartrate-resistant acid phos- To assess whether the increase in osteoclast differ- phatase (TRAP) þ cells (Figure 6a). entiation was mediated by IL-8 and/or IL-6, we treated To determine whether this effect was mediated by pro- the conditioned media derived from D140-overexpressing osteoclastogenic factors secreted in the conditioned media, A549 and H460 cells with IL-8 and IL-6 blocking we used enzyme-linked immunosorbent assay to measure antibodies or their combination. The conditioned media the levels of several factors previously implicated in was subsequently used to perform an in vitro osteoclas- osteoclastogenesis, including receptor-activated nuclear togenic assay. No differences were obtained in the area factor-k B-ligand, such as interleukin (IL)-6, tumor necrosis or the number of TRAP þ cells (data not shown). These factor-a, IL-8 (De Larco et al., 2001) and MIP-1a (Mundy, data suggest that IL-8 and/or IL-6 are not sufﬁcient to 2002). Interestingly, the pro-osteoclastogenic cytokine IL-8 mediate the pro-osteoclastogenic effects observed in was upregulated in the conditioned media from H460 and D140-overexpressing cells. A549 cells overexpressing D140.Furthermore,ADAM8 siRNA in both cell lines resulted in an increase in D140 expression together with an almost twofold increase in IL-8 The effects of ADAM8 and its isoforms in vivo protein secretion levels as compared with scramble siRNA- Previous in vitro studies have suggested that ADAM8 treated cells (Figure 6c). In contrast, only D140-over- could have a role in the acquisition of an aggressive expressing H460 cells secreted high IL-6 levels, whereas IL- phenotype (Ishikawa et al., 2004, Valkovskaya et al., 2007, 6 levels in A549 overexpressors were undetected. These Wildeboer et al., 2006). Thus, we examined the extent to differences were probably related to the different histolo- which the D140 isoform mediated these detrimental effects. gical origin of these tumor cell lines. No differences in IL- To this aim, we selected human adenocarcinoma A549 11, osteoprotegerin, receptor-activated nuclear factor-k B- cells, which represent the most frequent histological ligand and MIP-1a levels were observed (data not shown). subtype in NSCLC. By inoculation of lung cancer cells in These results indicate that the presence of D140 isoform athymic nude mice, this model induces rapid and leads to an increase in the pro-osteoclastogenic cytokine reproducible metastasis to long bones (data not shown). secretion, which may partially contribute to the enhanced When we inoculated ADAM8-overexpressing A549 osteoclast formation in vitro. cells into athymic nude mice, bone lesions were evident

Oncogene Mechanism of bone colonization I Herna´ndez et al 3764 2 m μ 15 20 100 * 15 ** 10 10 50 Control 5

survival (%) 5 Metastasis-free

* Δ14' Metastatic area/

0 total bone area (%) 0 0 010203040 Mock Δ14' Number of osteoclasts Mock Delta14

Time (Days) at tumor-bone interface / Mock Δ14´ Mock Δ14´ 21d 28d34d μCT H/E Mock

T T ´ 14 Δ

B B

Figure 7 In vivo metastatic assay. Cells overexpressing D140 isoform and mock-transduced were inoculated in the left cardiac ventricle of athymic nude mice. (a) Kaplan–Meier curve of the bone metastasis-free survival. (b) Computerized X-ray image analysis obtained 34 days after intracardiac inoculation of D140-overexpressing A549 cells and mock-transduced cells. Data are given as the mean±s.e.m. of eight mice per group; calculated using the rank-sum test with Bonferroni adjustment. (c) Representative images of long bones obtained at 21, 28 and 34 days after inoculation by X-ray imaging (left), three-dimensional reconstruction of mCT (middle) and hematoxylin and eosin histological staining (right). (d) Quantiﬁcation of the number of TRAP þ cells at tumor–bone interphase in long bones of inoculated animals with mock and D140-overexpressing A549 cells. (e) Representative TRAP þ histological sections of long bones from animals inoculated with mock and D140-overexpressing A549 cells (left panel). Detailed images (right panels) showing TRAP þ cells in purple (black arrows) at tumor (T)– bone (B) interphase in the subperiosteal region of the cortical bone (B). A more invasive front with high numbers of TRAP þ cells was observed in animals inoculated with D140 cells (Bar ¼ 50 mm). (*Po0.05, **Po0.01).

by X-ray 20 days after inoculation. No differences were In contrast, discrete focal colonies of tumoral cells were found in the number and area of the osteolytic lesions of found in long bones of control mice at 34 days after ADAM8-inoculated mice as compared with mock-trans- inoculation. To investigate whether these lesions were duced inoculated animals (data not shown). However, the caused by an increase in bone resorption, TRAP overall survival curve revealed a higher mortality in staining was carried out to reveal the presence of animals inoculated with ADAM8 compared with control osteoclasts. At the interface between the periosteum cells (data not shown). The cause of lethality could not be and the cortical bone, and consistent with previous determined from necropsy of the animals. However, findings, the number of multinucleated TRAP þ osteo- cachexia developed faster in ADAM8-inoculated animals clasts per bone interface was significantly increased in than in control mice. These findings suggest that an D140-inoculated animals (Po0.01) (Figure 7d). unknown direct or indirect effect mediated by ADAM8 These data suggest that both ADAM8 and its D140 may be critically affecting survival. isoform contribute to an aggressive tumor phenotype In contrast, when we inoculated D140-overexpressing in vivo. Moreover, D140 isoform expression results in the cells we found slight but significant decrease in the time faster development of aggressive osteolytic lesions, of appearance of bone metastatic lesions in this animals mediated by an increase in osteoclast number and as compared with control mice (Po0.05) (Figure 7a). higher invasive activity. These results were correlated with a significant increase in the area of osteolytic lesions over time, as assessed by X-ray image analysis and m-computerized tomography scan (Figures 7b and c) compared with mock-trans- Discussion duced inoculated animals. Histological analysis of long bones derived from D140 animals revealed a complete We have identified several novel truncated isoforms of colonization of the bone marrow compartment with ADAM8 derived from alternative splicing in lung cancer destruction of cortical bone in the metaphyseal region. cells and shown that their functional involvement in

Oncogene Mechanism of bone colonization I Hernańdez et al 3765 invasiveness and osteoclastogenesis contributes to their the MMP domain had a similar effect to the D140 aggressive phenotype. In a bone metastasis model of isoform, suggests that the MMP domain would be NSCLC in vivo, these isoforms cooperate to increase dispensable for the activity of D140 (Choi et al., 2001). osteolytic metastasis during the early stages of coloniza- Of note, the novel juxtaposed 14 amino-acid sequence tion leading to an increased osteolytic area of metas- remarkably contains several cysteines, which could tasis. Our findings unveil a novel mechanism by which preserve its functional activity. tumor cells are able to thrive in the bone compartment. Our data suggest that D140 activity could be mediated The identification of previously undescribed ADAM8 by the shedding or activation of secondary targets that isoforms represents a common feature of the ADAMs participate in osteoclastogenesis. Several findings sup- family. Previously, two phylogenetically related mem- port this contention. First, D140 upregulation in condi- bers, ADAM12 and ADAM28, that share an identical tions that mimic tumor–stroma interactions supports its exonic structure with ADAM8, have been shown to role in the cell–cell interactions. Second, the fact that generate truncated variants with a high degree of both forms were detected in cell lysates rather than in structural similarity (Gilpin et al., 1998, Haidl et al., conditioned media supports a putative role as a 2002). Both the D18 isoform of ADAM8 and the s12- sheddase or in integrin binding. Finally, we found that splicing variant of ADAM12 contain a frameshift a pro-osteoclastogenic cytokines IL-8 and IL-6 were insertion, generated by the partial intronic transcription upregulated in cells overexpressing the D140 isoform. that yields a truncated protein, which lacks a transmem- Interestingly, production of IL-8 was driven by ADAM- brane domain. Similarly, the ADAM28s isoform con- b1 integrin interactions in natural killer cells (Mainiero tains a novel translated sequence of 56 nucleotides in et al., 2000). On the basis of these findings, it is tempting intron 14 that results in a truncated protein with a 17 to speculate that the binding of the D140 isoform to b1- amino acid short cysteine-rich domain, remarkably integrin in tumor cells may activate downstream similar to the D140 isoform. These findings suggest a signaling events that lead to the subsequent release of similar evolutionary origin (Fourie et al., 2003) (Kim IL-8 and osteoclast formation. A similar mechanism for et al., 2006) and highly conserved mechanism of splicing IL-6 upregulation has been described in osteoblasts after among different ADAM family members. interactions between the ADAM9 disintegrin domain More importantly, ADAM8 truncated isoforms also and avb5 (Karadag et al., 2006). Although both IL-6 and showed distinct functions compared with the native IL-8 were elevated, their blockade was not sufficient to protein, as previously reported for other members of the decrease osteoclast differentiation in vitro, suggesting ADAMs family (Reiss et al., 2006, Seals and Court- that other unidentified pro-osteoclastogenic factors neidge, 2003). In agreement with these earlier studies, released in the conditioned media of D140-overexpres- the novel D140 isoform identified in this report showed a sing cells might account for this effect. marked increase in the induction of TRAP þ cells. Besides the integrin–ADAM interaction, the specific Previous studies have described a 65 kDa human soluble targets of the metalloproteinase domains remain elusive. isoform and a 70 kDa truncated murine isoform of in vitro studies have shown that ADAM8 can process ADAM8, structurally similar to the D140 isoform (Choi several molecules including CD40, tumor necrosis et al., 2001, Mandelin et al., 2003). This soluble murine factor-a, IL1R and the immunoglobulin E receptor ADAM8 isoform induced the formation of bone- CD23 (Fourie et al., 2003). Only the CHL1 neural-cell resorbing osteoclasts and acted at later stages of adhesion homolog (Naus et al., 2004), NCAM-140 osteoclast differentiation and precursor fusion (Choi (Hinkle et al., 2006) and L-selectin (Gomez-Gaviro et al., et al., 2001). The fact that this murine form was 2007) have been shown to be processed by ADAM8 structurally and functionally related to the D140 isoform, in vivo. Whether these or other unknown adhesion which also exhibited a similar pro-osteoclastogenic molecules can be targeted by ADAM8 during invasion effect, emphasizes the relevance of ADAM8 in osteo- remains to be addressed. Our findings support an clastogenesis (Choi et al., 2001, Mandelin et al., 2003, unambiguous role of full-length ADAM8 in the inva- Rao et al., 2006, Verrier et al., 2004). This effect on siveness of H460 and A549 cells, in agreement with osteoclast formation was mediated by the interactions previous findings (Ishikawa et al., 2004, Valkovskaya between the disintegrin domain and a9b1 integrin (Rao et al., 2007, Wildeboer et al., 2006). Although D18a et al., 2006). Indeed, D140 isoform lacks the hypervari- contributed to the increased invasiveness observed here, able region, so the intact disintegrin domain would be its role still remains unclear, especially as forced more readily available to interact with an integrin expression was associated with concomitant overexpres- molecule. In contrast, three-dimensional data suggest sion of the native protein. The proinvasive activity that the disintegrin domain in wild-type ADAM8 would induced by wild-type ADAM8 would require the be partially hidden by the hypervariable region (Takeda coordinated activity of cell-matrix adhesion and degra- et al., 2007). These structural differences could explain dation, cytoskeleton organization and motility. Previous the higher activity of the D140 isoform than the findings have suggested a role for ADAM12, which is full-length isoform in the fusion of mononuclear closely related to ADAM8, in the extracellular proces- precursors. Another critical domain for osteoclast sing of target substrates that favor motility and formation, the cysteine-rich domain, would be partially cytoskeletal remodeling (Kveiborg et al., 2008) such as preserved in the D140 isoform. However, an earlier study Src (Stautz et al., 2010), through interactions with key showing that a truncated isoform of ADAM8 lacking components of cell-matrix adhesion such as actinin-1

Oncogene Mechanism of bone colonization I Hernańdez et al 3766 (Cao et al., 2001) and actinin-2 (Galliano et al., 2000). Adam8- Integrin interactions Similarly, ADAM8 has been implicated in the proces- Δ18a sing of adhesion proteins in other systems (Hooper Δ14 and Lendeckel, 2005). Both membrane-anchored and 1 secreted forms of ADAM8 would participate in matrix adhesion (Schlomann et al., 2002), whereas other MMPs Vesicles could process the adhesive proteins resulting in migra- for extracellular tion (Gomez-Gaviro et al., 2007). A related mechanism 2 MMP release has been suggested for the processing of vascular cell adhesion molecule on eosinophils (Garton et al., 2003, Matsuno et al., 2007) and L-selectin on neutrophils (Gomez-Gaviro et al., 2007). Interestingly, ADAM8 was found in intracellular vesicles (Gomez-Gaviro et al., 2007, Verrier et al., 2004), a location associated with the cargo Shedase transport of MMP and other molecules to the extracellular Activity microenvironment, which would allow cell invasion. These in vitro findings were associated with the early 3 development in osteolytic lesions in the process of bone metastasis. We have recently shown in a model of Invasion Prosteoclastogenic NSCLC bone metastasis that tumor development in the factors bone compartment is mediated by two distinct mechanisms (Vicent et al., 2008). During the early stages, bone Figure 8 Hypothetical model of ADAM8-mediated colonization. ADAM8 and its spliced variants would allow tumor cells to thrive osteolysis is driven by tumor-induced osteoclastogen- in the bone compartment by a triple action. As a consequence of esis. In the later stages, tumor–stroma interactions perturbed splicing machinery, (1) the D140 isoform would bind to might be more significant in increasing metalloproteo- integrins including a9b1, thereby initiating a signaling cascade that lytic activity resulting in bone destruction and metastatic leads to increased secretion of pro-osteoclastogenic cytokines IL-8 and IL-6. Complementary, ADAM8 and its isoforms could growth, probably due to increased invasiveness and associate with MMPs in cargo vesicles modulating their extra- osteoclastogenesis. In this scenario, ADAM8-derived cellular transport and activity (2). Cytokine release into the isoforms would contribute to the early stages of extracellular milieu or direct shedding by ADAM8 MMP proces- metastatic development in vivo. Interestingly, over- sing would then contribute to increase osteoclastogenesis and 0 expression of ADAM8 protein was significantly more invasion (3). This increase induced by the D14 isoform would be modulated by complex tumor–stroma interactions and bone common in tumors from the patients with locally matrix-derived factors that would exacerbate invasiveness, osteo- advanced lung cancer or distant organ metastases than clastic activity leading to deleterious osseous lesions. those with earlier stage disease (Ishikawa et al., 2004). On the basis of their close similarity to ADAM8 as well In conclusion, we have identified novel truncated as their redundant roles shown in the other systems, forms of ADAM8 that were upregulated in tumor cells. other ADAMs may functionally cooperate with Characterization of their functional role in vitro revealed ADAM8. In this regard, the closely related isoform increased invasive abilities and osteoclastogenic proper- ADAM28s is overexpressed in the human NSCLCs and ties, and correlated with the early development of its high expression levels are correlated with lymph node metastatic lesions in vivo (Figure 8). In this context, metastasis (Ohtsuka et al., 2006). More importantly, these truncated isoforms that were upregulated by several ADAMs have been shown to mediate epithelial- tumor–stroma interactions could induce a high tumor derived growth factor receptor transactivation induced burden and tumor-induced osteolytic lesions, by a by G-protein-coupled receptors in various cells or mechanism that may involve IL-6 and IL-8. These tissues. ADAM10, ADAM12, ADAM15 and ADAM17 findings suggest a novel mechanism of bone colonization were able to shed epithelial-derived growth factor in lung cancer bone metastasis. receptor ligands including heparin binding epidermal growth factor, transforming growth factor-a and amphiregulin after cell stimulation with G-protein- coupled receptor agonists such as angiotensin II, Materials and methods lipopolysaccharide, IL-8, lipoteichioc acid, phenylephr- ine, carbachol or bombesin, leading to activation of Cell lines and culture conditions epithelial-derived growth factor receptor-mediated sig- ST-2 and a battery of lung cancer cell lines were obtained from naling pathways (Blobel, 2005, Gee and Knowlden, the ATCC (Manassas, VA, USA). Lung cancer cells lines 2003, Higashiyama and Nanba, 2005, Ohtsu et al., were grown in RPMI 1640 with L-glutamine (Invitrogen, Barcelona, Spain) supplemented with 10% fetal bovine serum 2006). These data emphasize the fact that global (Invitrogen), 1% penicillin and streptomycin (Invitrogen). Cell inhibition of ADAMs may be highly beneficial in the lines included represent a variety of lung histological subtypes: development of antimetastatic therapies. Indeed, a SCLC (H69, H209, H345, H510, H82 and H187), adenocarci- broad MMP/ADAM inhibitor GM6001 was found to nomas (H23 and H2087), bronchioalveolar carcinomas be effective in inhibiting bone metastasis progression in (A549), large cell carcinomas (H460 and H661), squamous a NSCLC animal model (Vicent et al., 2008). cell carcinomas (H157), and carcinoid tumor (H727). Normal

Oncogene Mechanism of bone colonization I Hernańdez et al 3767 bronchioalveolar primary cells were also used: NHBE lysates for subsequent protein and RNA extraction. Other (Clonetics, Lonza, Cologne, Germany) was grown in BECGM methods are included as supplementary information. Systems (Cambrex, Karlskoga, Sweden). ST-2 murine stromal cells were grown using Dulbecco’s modified Eagle’s medium In vivo assays with ultraglutamine (Cambrex). All protocols were approved by the local Committee on Animal Research and Ethics. Female athymic nude mice, 3 ADAM8 antibodies weeks old (eight animals per group), were inoculated with A8-MD is a polyclonal rabbit antibody against a peptide different cells. Mice were killed 28 days after inoculation as partially, overlapping the catalytic and disintegrin domains of previously described (Gonzalez et al., 2007). human ADAM8 (ab11527, Abcam, Cambridge, UK). a-b-Actin was a monoclonal anti-b-actin (clone AC-15, Sigma, St Louis, Invasion assays MO, USA). Invasion assays were carried out with conditioned medium from murine bone marrow stromal ST-2 cells as chemoattractant placed in the lower compartment of 8-mm pore Boyden chamber. Cloning of human ADAM8 and isoforms Cells (2 Â 105) were seeded in each well, in sixplicate for each The primer sequences used for the screening were as follows: condition. The upper chamber was precoated with 420 ng 50-ATGTGTGACCTCGAGGAGTTCT-30 and 50-GGATGT 0 matrigel per ml (Sigma) dried at room temperature for 5 h. Cells CATAGGAGAAGCAGGA-3 . Primer sequences used for 4 0 (5 Â 10 ) were seeded on the upper compartment in serum-free real-time PCR were as follows: 5 -GGACCAGAAGCGGGT medium per well in 24-well plates (Corning Life Sciences, TAG-30 and 50-AGGTGAAGTGGGCACAGAG-30 for D140 0 0 Chorges, France). After 24 h, cells on the top chamber were and primers 5 -TGACTGAGGTGCACGCAGCCC-3 and wiped with a cotton swab, and cells in the lower compartment 50-GGCTCCCGGACGCTTACACTG-30 for D18a. The primer 0 were fixed and stained with crystal violet. Number of invasive sequences used for cloning were as follows: 5 -GCAGGAACC cells was evaluated with a computerized image analysis system, AGACCGTGT-30 (50 untranslated region sequence) and 50-GA AnalySIS (Mu¨nster, Germany). TGCATTACTGAGGTTAGAACAGC-30 (30 untranslated region sequence) for full ADAM8 (fragment of 2680 bp expected); Statistical analysis Previous 50 untranslated region sequence and 50-TGTGGGACC AGAAGCGGGTTA-30 (intron 14 sequence) for truncated To study differences in invasion activity and osteoclastogenic ADAM8 (fragment of 1689 bp expected). The PCR conditions activity, data were analyzed by one-way analysis of variance, post hoc were 94 1Cfor5min;941C for 30 s, 60 1C for 45 s, 68 1Cfor and Bonferroni test. SPSS software (Chicago, IL, 3 min for 40 cycles and a final extension at 68 1C for 10 min. USA) was used for the analysis. Values were expressed as ± P Selected fragments were extracted with Gel Extraction Kit means s.d. and statistical significance was defined as o0.05 P P (Qiagen, Hilden, Germany) cloned in the pCR Blunt II TOPO (*), o0.01 (**), o0.001 (***). To analyze the results of in vivo U (Invitrogen) and sequenced to verify insert fidelity and integrity. assays, the one-tailed Mann–Whitney -test for nonparametric samples was used.

Transfections and knockdown Insert complementary DNAs for wild-type ADAM8 , D18a Abbreviations and D140 were excised and subcloned in retroviral vector, pBabe-Neo (Addgene, Cambridge, MA, USA), After infection of the cell lines H460 and A549, antibiotic-resistant pools were MMP, metalloprotease; NSCLC, nonsmall cell lung cancer; expanded and frozen at first passages. Cells were screened by SCLC, small cell lung cancer; TRAP, tartrate-resistant acid western blotting to assess expression levels after transduction. phosphatase. For knockdown experiments, short hairpin RNAi constructs were cloned into pSilencer 4.1-CMV neo (Ambion, Austin, TX, USA). Transfection was carried out with Lipofectamine Conflict of interest 2000 (Invitrogen) method in both cell lines. The pSilencer 4.1- CMV neo with a scramble sequence was used as mock. The authors declare no conflict of interest. Geneticin-resistant clones that stably express the short hairpin RNAis were expanded and frozen at first passages. The sequences that resulted in an effective knockdown were as Acknowledgements follows: ADAM8–1999: 50-AGGACGTTGCCAGGACTT 0 0 ACA-3 (siRNA1); ADAM8–2221: 5 -AGGCATCATCGTCT We thank especially S Martıńez and the members of the ACCGCAA-3 (siRNA2). Morphology and Animal Core Facilities. C Berasain for critical reading of the article. This work was supported by Coculture conditions ‘UTE project FIMA’ agreement, and RTICCC C03/10, FIT- A total of 500 000 ST2 cells were seeded with or without 090100-2005-46, PI042284, PI070031 and SAF-2009-11280 (to 1 Â 106 H460 or A549 cells and grown for 3 days. Conditioned FL). FL is also supported by funds from the I3 Program, ‘La media obtained after 24 h incubation with serum-free medium Caixa Foundation’, and is a recipient of the ‘Ortiz de under these coculture conditions was collected together with Landa´zuri’ award (67/2005, Government of Navarra).

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