Title: ADAM12 Transmembrane and Secreted Isoforms Promote Breast Tumor Growth
Abstract: Increased levels of ADAM12 have been reported in a variety of human cancers. We have previously reported that urinary ADAM12 is predictive of disease status in breast cancer patients and that ADAM12 protein levels in urine increase with progression of disease. On the basis of these findings, the goal of this study was to elucidate the contribution of ADAM12 in breast tumor growth and progression. Overexpression of both the ADAM12-L (transmembrane) and ADAM12-S (secreted) isoforms in human breast tumor cells resulted in a significantly higher rate of tumor take and increased tumor size. Cells expressing the enzymatically inactive form of the secreted isoform, ADAM12-S, had tumor take rates and tumor volumes similar to those of wild-type cells, suggesting that the tumor-promoting activity of ADAM12-S was a function of its proteolytic activity. Of the two isoforms, only the secreted isoform, ADAM12-S, enhanced the ability of tumor cells to migrate and invade in vitro and resulted in a higher incidence of local and distant metastasis in vivo. This stimulatory effect of ADAM12-S on migration and invasion was dependent on its catalytic activity. Expression of both ADAM12 isoforms was found to be significantly elevated in human malignant breast tissue. Taken together, our results suggest that ADAM12 overexpression results in increased tumor take, tumor size, and metastasis in vivo. These findings suggest that ADAM12 may represent a potential therapeutic target in breast cancer. Increased levels of ADAM12 have been reported in a variety of human cancers. We have previously reported that urinary ADAM12 is predictive of disease status in breast cancer patients and that ADAM12 protein levels in urine increase with progression of disease. On the basis of these findings, the goal of this study was to elucidate the contribution of ADAM12 in breast tumor growth and progression. Overexpression of both the ADAM12-L (transmembrane) and ADAM12-S (secreted) isoforms in human breast tumor cells resulted in a significantly higher rate of tumor take and increased tumor size. Cells expressing the enzymatically inactive form of the secreted isoform, ADAM12-S, had tumor take rates and tumor volumes similar to those of wild-type cells, suggesting that the tumor-promoting activity of ADAM12-S was a function of its proteolytic activity. Of the two isoforms, only the secreted isoform, ADAM12-S, enhanced the ability of tumor cells to migrate and invade in vitro and resulted in a higher incidence of local and distant metastasis in vivo. This stimulatory effect of ADAM12-S on migration and invasion was dependent on its catalytic activity. Expression of both ADAM12 isoforms was found to be significantly elevated in human malignant breast tissue. Taken together, our results suggest that ADAM12 overexpression results in increased tumor take, tumor size, and metastasis in vivo. These findings suggest that ADAM12 may represent a potential therapeutic target in breast cancer. IntroductionHuman ADAM12 (meltrin α, MCMP) is expressed as two alternatively spliced forms, a membrane-anchored long form (ADAM12-L) and a short secreted form (ADAM12-S) (1Gilpin B.J. Loechel F. Mattei M.G. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). ADAM12-L and -S share a high overall sequence homology, differing only in the transmembrane domain (that ADAM12-S lacks) and a C terminus that is distinct in each isoform. Discrete functions can be attributed to the individual ADAM12 domains. The catalytic domain of ADAM12 contains the consensus HEXGHXXGXXHD zinc-binding motif, and both isoforms are active proteases. ADAM12-L sheds several membrane-bound ligands, including heparin-binding EGF-like growth factor binding protein (HB-EGF) (2Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (639) Google Scholar), EGF (3Horiuchi K. Zhou H.M. Kelly K. Manova K. Blobel C.P. Dev. Biol. 2005; 283: 459-471Crossref PubMed Scopus (143) Google Scholar), betacellulin (3Horiuchi K. Zhou H.M. Kelly K. Manova K. Blobel C.P. Dev. Biol. 2005; 283: 459-471Crossref PubMed Scopus (143) Google Scholar), Notch ligand delta-like 1 (4Dyczynska E. Sun D. Yi H. Sehara-Fujisawa A. Blobel C.P. Zolkiewska A. J. Biol. Chem. 2007; 282: 436-444Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and placental leucine aminopeptidase (5Ito N. Nomura S. Iwase A. Ito T. Kikkawa F. Tsujimoto M. Ishiura S. Mizutani S. Biochem. Biophys. Res. Commun. 2004; 314: 1008-1013Crossref PubMed Scopus (76) Google Scholar). ADAM12-S can cleave insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 (6Loechel F. Gilpin B.J. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 16993-16997Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 7Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (276) Google Scholar) and degrade extracellular matrix substrates (8Roy R. Wewer U.M. Zurakowski D. Pories S.E. Moses M.A. J. Biol. Chem. 2004; 279: 51323-51330Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). ADAM12 mRNA and protein are highly expressed in a variety of malignant tumor tissues and tumor cell lines including breast, brain, bladder, gastric, colon, lung, laryngeal, and hepatocellular carcinomas (for a review, see Refs. 9Kveiborg M. Albrechtsen R. Couchman J.R. Wewer U.M. Int. J. Biochem. Cell Biol. 2008; 40: 1685-1702Crossref PubMed Scopus (138) Google Scholar, 10Lendeckel U. Kohl J. Arndt M. Carl-McGrath S. Donat H. Röcken C. J. Cancer Res. Clin. Oncol. 2005; 131: 41-48Crossref PubMed Scopus (133) Google Scholar, 11Fröhlich C. Albrechtsen R. Dyrskjøt L. Rudkjaer L. Ørntoft T.F. Wewer U.M. Clin. Cancer Res. 2006; 12: 7359-7368Crossref PubMed Scopus (103) Google Scholar, 12Kodama T. Ikeda E. Okada A. Ohtsuka T. Shimoda M. Shiomi T. Yoshida K. Nakada M. Ohuchi E. Okada Y. Am. J. Pathol. 2004; 165: 1743-1753Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Carl-McGrath S. Lendeckel U. Ebert M. Roessner A. Röcken C. Int. J. Oncol. 2005; 26: 17-24PubMed Google Scholar, 14Markowski J. Oczko-Wojciechowska M. Gierek T. Jarzab M. Paluch J. Kowalska M. Wygoda Z. Pfeifer A. Tyszkiewicz T. Jarzab B. Niedzielska I. Borgiel-Marek H. J. Physiol. Pharmacol. 2009; 60: 57-63PubMed Google Scholar, 15Mino N. Miyahara R. Nakayama E. Takahashi T. Takahashi A. Iwakiri S. Sonobe M. Okubo K. Hirata T. Sehara A. Date H. J. Surg. Oncol. 2009; 100: 267-272Crossref PubMed Scopus (35) Google Scholar, 16Le Pabic H. Bonnier D. Wewer U.M. Coutand A. Musso O. Baffet G. Clément B. Théret N. Hepatology. 2003; 37: 1056-1066Crossref PubMed Scopus (181) Google Scholar). The role of the two isoforms of ADAM12 or mechanistic studies describing their functional role in cancer have been rare to date. In this report, we have utilized an in vivo orthotopic tumor model that reliably recapitulates human breast tumor growth to investigate the role of the two distinct isoforms of ADAM12 in the development of invasive breast cancer.ADAM12 can be detected in the urine of breast (8Roy R. Wewer U.M. Zurakowski D. Pories S.E. Moses M.A. J. Biol. Chem. 2004; 279: 51323-51330Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and bladder (11Fröhlich C. Albrechtsen R. Dyrskjøt L. Rudkjaer L. Ørntoft T.F. Wewer U.M. Clin. Cancer Res. 2006; 12: 7359-7368Crossref PubMed Scopus (103) Google Scholar) cancer patients, and its levels have been shown to correlate with disease status, stage, and cancer risk (8Roy R. Wewer U.M. Zurakowski D. Pories S.E. Moses M.A. J. Biol. Chem. 2004; 279: 51323-51330Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 17Pories S.E. Zurakowski D. Roy R. Lamb C.C. Raza S. Exarhopoulos A. Scheib R.G. Schumer S. Lenahan C. Borges V. Louis G.W. Anand A. Isakovich N. Hirshfield-Bartek J. Wewer U. Lotz M.M. Moses M.A. Cancer Epidemiol. Biomark. Prev. 2008; 17: 1034-1042Crossref PubMed Scopus (79) Google Scholar). The discovery of ADAM12 as a potential biomarker for breast cancer begged the question of its relevance in human breast tumorigenesis. We show that overexpression of both ADAM12 isoforms in breast tumor cells promotes tumor growth and that only ADAM12-S expression stimulates their migration and invasion in vitro and local and distant invasion in vivo as a function of its proteolytic activity. We further show that both ADAM12-L and ADAM12-S expression levels are significantly higher in human malignant breast tissue and metastatic lymph nodes compared with normal breast tissue.DISCUSSIONWe have previously reported that urinary ADAM12 is predictive of disease status in breast cancer patients and that its levels in urine increase with progression of the disease. Increased levels of ADAM12 mRNA and protein have previously been reported in a number of cancers and tumor cell lines, suggesting an active role for ADAM12 in tumor progression. However, these studies did not identify either the causal role or the distinct contributions of either ADAM12 isoform in tumorigenesis. This study is the only one to date to describe a role for ADAM12 overexpression in human cancer cells using an orthotopic breast tumor model.IHC of a panel human breast tumors indicated that ADAM12 expression is up-regulated in primary tumors at early stages and is maintained at later stages of tumorigenesis and in metastatic LN. Of the matched sets of primary tumor tissue and metastatic LN analyzed in this study, a majority had positive expression of ADAM12 in both the primary tumor and matched LN, suggesting that ADAM12 expression may be essential for primary tumor growth as well as promoting and/or maintaining distant metastasis. Interestingly, within this cohort, ∼13% of the primary tumors did not stain positive for ADAM12, whereas their matched LNs did (Fig. 1D), suggesting that metastatic cells originating from primary tumors expressing low levels or no ADAM12 may up-regulate ADAM12 expression during the process of invasion and that ADAM12 function may be critical for the establishment of distant metastasis.This is the first report to analyze expression of both the transmembrane and secreted isoforms of ADAM12 in human breast cancer. Our data indicate that there is increased expression of both isoforms of ADAM12 in human breast cancer tissue. Interestingly, expression of the transmembrane isoform, ADAM12-L, was found to be slightly but significantly higher in early stages of breast tumorigenesis, whereas both forms are expressed in late-stage disease. The tumor stage-dependent differences in the expression levels of ADAM12 isoforms may result in their distinct contributions to the tumor growth and metastasis process. Increased expression of the transmembrane isoform, ADAM12-L, in Stage I and II breast cancer may reflect its role in promoting tumor growth, perhaps via its sheddase and growth factor receptor activation function. The high levels of ADAM12-S in late-stage human breast tumors support its potential role in tumor invasion and metastasis.ADAM12 expression and catalytic function has not previously been correlated with enhanced tumor cell proliferation, although its effect on the proliferation of normal cells has been reported. ADAM12-L was reported to regulate bronchial epithelial cell proliferation and apoptosis in an HB-EGF- and EGFR-dependent manner (22Rocks N. Estrella C. Paulissen G. Quesada-Calvo F. Gilles C. Guéders M.M. Crahay C. Foidart J.M. Gosset P. Noel A. Cataldo D.D. Cell Prolif. 2008; 41: 988-1001Crossref PubMed Scopus (27) Google Scholar). In addition, ADAM12-L has been reported to enhance chondrocyte proliferation in osteoarthritis via the degradation of IGFBP-5 and increased bioavailability of insulin-like growth factor (23Okada A. Mochizuki S. Yatabe T. Kimura T. Shiomi T. Fujita Y. Matsumoto H. Sehara-Fujisawa A. Iwamoto Y. Okada Y. Arthritis Rheum. 2008; 58: 778-789Crossref PubMed Scopus (65) Google Scholar). Therefore, an increased expression of ADAM12-L in early-stage breast cancer, coupled with its unique sheddase function, may suggest a role for this protease in breast tumor growth in vivo. The secreted isoform, ADAM12-S, which is rarely expressed in normal adult tissue (placenta is an exception) and which we found to promote tumor cell migration and invasion in vitro and local and distant metastasis in vivo, was expressed in late stage human breast tumors, indicating a potential role in tumor invasion and metastasis. Surprisingly, ADAM12 expression in human breast tumors appeared to be primarily epithelial rather than stromal. This is in contrast to mouse models of breast and prostate tumors, where ADAM12 staining was detected almost exclusively in the stroma (24Peduto L. Reuter V.E. Sehara-Fujisawa A. Shaffer D.R. Scher H.I. Blobel C.P. Oncogene. 2006; 25: 5462-5466Crossref PubMed Scopus (86) Google Scholar). This difference suggests that the ADAM12-overexpressing orthotopic breast tumor model, used in this study, may reliably recapitulate human breast tumor growth compared with other models.Two recent studies have begun to investigate a role for ADAM12 in tumorigenesis using transgenic mouse models. One report used the mouse mammary tumor virus-polyomavirus middle T oncogene (MMTV-PyMT) mouse breast tumor model to show that ADAM12 transcripts were strongly up-regulated in the stromal cell subpopulation adjacent to epithelial tumor cells, which were largely negative for ADAM12 expression (24Peduto L. Reuter V.E. Sehara-Fujisawa A. Shaffer D.R. Scher H.I. Blobel C.P. Oncogene. 2006; 25: 5462-5466Crossref PubMed Scopus (86) Google Scholar). In a gain of function study, transgenic overexpression of human ADAM12 in MMTV-PyMT mice was reported to accelerate breast tumor progression and increase tumor burden and aggressiveness (25Kveiborg M. Fröhlich C. Albrechtsen R. Tischler V. Dietrich N. Holck P. Kronqvist P. Rank F. Mercurio A.M. Wewer U.M. Cancer Res. 2005; 65: 4754-4761Crossref PubMed Scopus (121) Google Scholar). This report attributed the effect of Adam 12 on tumor growth to its opposing effects on apoptosis (i.e. ADAM12 expression increased sensitivity of stromal cells to apoptosis but rendered tumor cells resistant to apoptosis). However, this observed effect of ADAM12 on apoptosis was independent of its proteolytic function (25Kveiborg M. Fröhlich C. Albrechtsen R. Tischler V. Dietrich N. Holck P. Kronqvist P. Rank F. Mercurio A.M. Wewer U.M. Cancer Res. 2005; 65: 4754-4761Crossref PubMed Scopus (121) Google Scholar). Although we observe a similar decrease in apoptotic rates in ADAM12-expressing breast tumor cells, our results are in contrast to the previous study in that our data indicate that catalytic activity of the secreted isoform, ADAM12-S, is essential for its protumorigenic and prometastatic effects in vitro as well as in vivo. In the case of the transmembrane isoform, we cannot rule out the possibility that the cysteine-rich, disintegrin or cytoplasmic domains of ADAM12-L may independently influence its tumorigenic potential. It is widely appreciated that use of the MMTV-PyMT model results in rapid development of multifocal mammary adenocarcinomas as well as local and distant metastasis, regardless of the expression of the gene/protein of interest, making it difficult to rigorously and fairly relegate any effects observed specifically to ADAM12. To address this, we have tested the effects of ADAM12 overexpression in the breast tumor epithelium in a less aggressive tumor model, which provides the opportunity to more clearly assess the potential role of this protease in the initial establishment of the human breast tumors (tumor take), growth, and metastasis. Using human cancer cells in an in vivo orthotopic breast tumor model, we demonstrate that ADAM12 expression significantly increased tumor take and tumor growth.We have recently reported that ADAM12 expression in breast tumor cells results in up-regulation of alternative growth pathways (18Roy R. Moses M.A. Breast Cancer Res. Treat. 2011; (in press)Google Scholar). Expression of ADAM12-L resulted in increased amphiregulin shedding and a concomitant up-regulation of EGFR protein expression and activation, ultimately resulting in estrogen-independent breast tumor cell proliferation. Similarly, increased ADAM12-S expression resulted in up-regulation of pIGF-1R and pMAPK levels in breast tumor cells (18Roy R. Moses M.A. Breast Cancer Res. Treat. 2011; (in press)Google Scholar). Therefore, one might speculate that ADAM12-induced higher tumor growth rates observed in this study may be associated with its effect on the up-regulation of alternative growth pathways. In the case of the secreted isoform, ADAM12-S, the tumor-promoting activity was a function of its catalytic function because cells expressing the enzymatically inactive form of the protease had tumor take rates and tumor volumes similar to parental MCF-7 cells.Comparative analysis of ADAM12 mRNA in breast cancer cell lines indicated that expression is significantly elevated in highly invasive, estrogen-receptor-negative lines such as MDA-MB-231 and Hs578T as compared with low metastatic, estrogen-responsive lines such as MCF-7 and T47-D. To begin to understand the contribution of the distinct isoforms of ADAM12 in promoting breast tumorigenesis, we established stable clones that overexpress ADAM12-L, ADAM12-S, and ADAM12-Scatmut, respectively, in MCF-7 cells. Expression of ADAM12-S resulted in increased migration and invasion of MCF-7 and T47-D breast tumor cells. This effect was specific for ADAM12-S, as silencing of expression led to a reversal of increased migration. Therefore, the metalloprotease function of ADAM12-S is crucial for the stimulation of migration because MCF-7 cells expressing the catalytically inactive form of the protein do not display increased migration in vitro. Interestingly, ADAM12-L-expressing cells did not display a similar increase in migratory capacity. This effect was recapitulated in the in vivo tumor studies. ADAM12-S tumors had increased local invasion of the surrounding area, a higher incidence of vascular invasion, a significantly higher incidence of LN metastasis (40%), as well as lung colonization of individual tumor cells (38%) as compared with WT MCF-7 tumors that displayed no LN or lung colonization. The ADAM12-l-expressing tumors were moderately invasive (LN metastasis, 21% and lung colonies, 20%). However, these rates were not found to be significantly higher than WT MCF-7-tumor-bearing animals. Our data suggests that expression of the secreted isoform, ADAM12-S, but not the transmembrane isoform, ADAM12-L, selectively increased local tumor invasion and incidence of vascular invasion and promoted significantly higher incidence of LN metastasis as well as lung colonization as compared with WT MCF-7 tumors. Interestingly, ADAM12 appears to exert distinct and opposing effects on tumor versus normal cell migration. For example, ADAM12 was reported to inhibit Chinese hamster ovary cell migration, although this activity was independent of its catalytic function (26Huang J. Bridges L.C. White J.M. Mol. Biol. Cell. 2005; 16: 4982-4991Crossref PubMed Scopus (91) Google Scholar). Similarly, ADAM12-deficient keratinocytes migrated significantly more than wild-type keratinocytes in a wound-healing mouse model (27Harsha A. Stojadinovic O. Brem H. Sehara-Fujisawa A. Wewer U. Loomis C.A. Blobel C.P. Tomic-Canic M. J. Mol. Med. 2008; 86: 961-969Crossref PubMed Scopus (41) Google Scholar). Importantly, both of these studies were conducted with normal cells, and neither of these studies explored isoform-specific effects on cell migration. Our results suggest that the secreted isoform, ADAM12-S, enhances the migratory and invasive capacity of breast tumor cells, thereby allowing malignant cells to locally invade as well as to form distant metastasis. Therefore, in normal tissue, ADAM12 may inhibit cell migration by mediating cell adhesion via integrin interactions, whereas, in tumor cells, higher levels of ADAM12-S proteolytic function stimulate cell migration and invasion. Although we cannot rule out the possibility that the effect of the secreted isoform, ADAM12-S, on tumor cell migration and invasion as well as tumor metastasis is mediated via integrin interactions, it has been previously reported that several ADAM family members can cleave extracellular matrix proteins (28Roy R. Zhang B. Moses M.A. Exp. Cell Res. 2006; 312: 608-622Crossref PubMed Scopus (157) Google Scholar). Therefore, given that the effect of ADAM12-S on tumor cell migration and invasion is dependent on catalytic function, it is probable that ADAM12-S stimulates migration and invasion via ECM cleavage and remodeling. Experiments exploring the mechanism by which ADAM12 may exert its effects of tumor proliferation and invasion are currently being conducted.Our study establishes distinct roles for ADAM12 isoforms in breast tumor progression and in local and distant tumor metastasis. These findings reflect the increased expression of ADAM12 in human breast cancer and metastatic lymph nodes and highlight the therapeutic potential of down-regulating or targeting ADAM12 in breast cancer, perhaps through the development of ADAM12-specific inhibitors (29Kveiborg M. Jacobsen J. Lee M.H. Nagase H. Wewer U.M. Murphy G. Biochem. J. 2010; 430: 79-86Crossref PubMed Scopus (33) Google Scholar). IntroductionHuman ADAM12 (meltrin α, MCMP) is expressed as two alternatively spliced forms, a membrane-anchored long form (ADAM12-L) and a short secreted form (ADAM12-S) (1Gilpin B.J. Loechel F. Mattei M.G. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). ADAM12-L and -S share a high overall sequence homology, differing only in the transmembrane domain (that ADAM12-S lacks) and a C terminus that is distinct in each isoform. Discrete functions can be attributed to the individual ADAM12 domains. The catalytic domain of ADAM12 contains the consensus HEXGHXXGXXHD zinc-binding motif, and both isoforms are active proteases. ADAM12-L sheds several membrane-bound ligands, including heparin-binding EGF-like growth factor binding protein (HB-EGF) (2Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (639) Google Scholar), EGF (3Horiuchi K. Zhou H.M. Kelly K. Manova K. Blobel C.P. Dev. Biol. 2005; 283: 459-471Crossref PubMed Scopus (143) Google Scholar), betacellulin (3Horiuchi K. Zhou H.M. Kelly K. Manova K. Blobel C.P. Dev. Biol. 2005; 283: 459-471Crossref PubMed Scopus (143) Google Scholar), Notch ligand delta-like 1 (4Dyczynska E. Sun D. Yi H. Sehara-Fujisawa A. Blobel C.P. Zolkiewska A. J. Biol. Chem. 2007; 282: 436-444Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and placental leucine aminopeptidase (5Ito N. Nomura S. Iwase A. Ito T. Kikkawa F. Tsujimoto M. Ishiura S. Mizutani S. Biochem. Biophys. Res. Commun. 2004; 314: 1008-1013Crossref PubMed Scopus (76) Google Scholar). ADAM12-S can cleave insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 (6Loechel F. Gilpin B.J. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 16993-16997Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 7Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (276) Google Scholar) and degrade extracellular matrix substrates (8Roy R. Wewer U.M. Zurakowski D. Pories S.E. Moses M.A. J. Biol. Chem. 2004; 279: 51323-51330Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). ADAM12 mRNA and protein are highly expressed in a variety of malignant tumor tissues and tumor cell lines including breast, brain, bladder, gastric, colon, lung, laryngeal, and hepatocellular carcinomas (for a review, see Refs. 9Kveiborg M. Albrechtsen R. Couchman J.R. Wewer U.M. Int. J. Biochem. Cell Biol. 2008; 40: 1685-1702Crossref PubMed Scopus (138) Google Scholar, 10Lendeckel U. Kohl J. Arndt M. Carl-McGrath S. Donat H. Röcken C. J. Cancer Res. Clin. Oncol. 2005; 131: 41-48Crossref PubMed Scopus (133) Google Scholar, 11Fröhlich C. Albrechtsen R. Dyrskjøt L. Rudkjaer L. Ørntoft T.F. Wewer U.M. Clin. Cancer Res. 2006; 12: 7359-7368Crossref PubMed Scopus (103) Google Scholar, 12Kodama T. Ikeda E. Okada A. Ohtsuka T. Shimoda M. Shiomi T. Yoshida K. Nakada M. Ohuchi E. Okada Y. Am. J. Pathol. 2004; 165: 1743-1753Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Carl-McGrath S. Lendeckel U. Ebert M. Roessner A. Röcken C. Int. J. Oncol. 2005; 26: 17-24PubMed Google Scholar, 14Markowski J. Oczko-Wojciechowska M. Gierek T. Jarzab M. Paluch J. Kowalska M. Wygoda Z. Pfeifer A. Tyszkiewicz T. Jarzab B. Niedzielska I. Borgiel-Marek H. J. Physiol. Pharmacol. 2009; 60: 57-63PubMed Google Scholar, 15Mino N. Miyahara R. Nakayama E. Takahashi T. Takahashi A. Iwakiri S. Sonobe M. Okubo K. Hirata T. Sehara A. Date H. J. Surg. Oncol. 2009; 100: 267-272Crossref PubMed Scopus (35) Google Scholar, 16Le Pabic H. Bonnier D. Wewer U.M. Coutand A. Musso O. Baffet G. Clément B. Théret N. Hepatology. 2003; 37: 1056-1066Crossref PubMed Scopus (181) Google Scholar). The role of the two isoforms of ADAM12 or mechanistic studies describing their functional role in cancer have been rare to date. In this report, we have utilized an in vivo orthotopic tumor model that reliably recapitulates human breast tumor growth to investigate the role of the two distinct isoforms of ADAM12 in the development of invasive breast cancer.ADAM12 can be detected in the urine of breast (8Roy R. Wewer U.M. Zurakowski D. Pories S.E. Moses M.A. J. Biol. Chem. 2004; 279: 51323-51330Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and bladder (11Fröhlich C. Albrechtsen R. Dyrskjøt L. Rudkjaer L. Ørntoft T.F. Wewer U.M. Clin. Cancer Res. 2006; 12: 7359-7368Crossref PubMed Scopus (103) Google Scholar) cancer patients, and its levels have been shown to correlate with disease status, stage, and cancer risk (8Roy R. Wewer U.M. Zurakowski D. Pories S.E. Moses M.A. J. Biol. Chem. 2004; 279: 51323-51330Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 17Pories S.E. Zurakowski D. Roy R. Lamb C.C. Raza S. Exarhopoulos A. Scheib R.G. Schumer S. Lenahan C. Borges V. Louis G.W. Anand A. Isakovich N. Hirshfield-Bartek J. Wewer U. Lotz M.M. Moses M.A. Cancer Epidemiol. Biomark. Prev. 2008; 17: 1034-1042Crossref PubMed Scopus (79) Google Scholar). The discovery of ADAM12 as a potential biomarker for breast cancer begged the question of its relevance in human breast tumorigenesis. We show that overexpression of both ADAM12 isoforms in breast tumor cells promotes tumor growth and that only ADAM12-S expression stimulates their migration and invasion in vitro and local and distant invasion in vivo as a function of its proteolytic activity. We further show that both ADAM12-L and ADAM12-S expression levels are significantly higher in human malignant breast tissue and metastatic lymph nodes compared with normal breast tissue.