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 Herna´ndez 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