ADAM9 Promotes Lung Cancer Metastases to Brain by a Plasminogen Activator-Based Pathway

Published OnlineFirst July 24, 2014; DOI: 10.1158/0008-5472.CAN-13-2995

Cancer Tumor and Stem Cell Biology Research

Chen-Yuan Lin1,2, Hung-Jen Chen3, Cheng-Chung Huang4, Liang-Chuan Lai5, Tzu-Pin Lu6, Guan-Chin Tseng7, Ting-Ting Kuo4, Qian-Yu Kuok4, Jennifer L. Hsu4,8,9, Shian-Ying Sung10, Mien-Chie Hung4,8,9,11, and Yuh-Pyng Sher1,4

Abstract The transmembrane cell adhesion protein ADAM9 has been implicated in cancer cell migration and lung cancer metastasis to the brain, but the underpinning mechanisms are unclear and clinical support has been lacking. Here, we demonstrate that ADAM9 enhances the ability of tissue plasminogen activator (tPA) to cleave and stimulate the function of the promigratory protein CDCP1 to promote lung metastasis. Blocking this mechanism of cancer cell migration prolonged survival in tumor-bearing mice and cooperated with dexamethasone and dasatinib (a dual Src/Abl kinase inhibitor) treatment to enhance cytotoxic treatment. In clinical specimens, high levels of ADAM9 and CDCP1 correlated with poor prognosis and high risk of mortality in patients with lung cancer. Moreover, ADAM9 levels in brain metastases derived from lung tumors were relatively higher than the levels observed in primary lung tumors. Our results show how ADAM9 regulates lung cancer metastasis to the brain by facilitating the tPA-mediated cleavage of CDCP1, with potential implications to target this network as a strategy to prevent or treat brain metastatic disease. Cancer Res; 74(18); 5229–43. 2014 AACR.

Introduction prognosis (2). Lung cancer accounts for the majority of brain Lung cancer is the leading cause of cancer-related deaths metastases. In contrast to metastases from other primary worldwide among both men and women, with 159,480 deaths cancer sites such as breast cancer, colorectal cancer, and estimated for 2013 in the United States (1). Brain metastases melanoma that can develop over 1 year after diagnosis, brain from lung are found in 20% to 50% of patients with non–small metastases from lung cancer can occur within months after or cell lung cancer (NSCLC) and cause the patients a worse even at the time of diagnosis of the primary (3). Once cancer cells intravasate from the primary site into the blood and before extravasation and colonization in the distant organ, the ﬁrst step for cancer cells to complete the process of 1Graduate Institute of Clinical Medical Science, China Medical University, metastasis is their adhesion to vascular endothelial cells (4). Taichung, Taiwan. 2Division of Hematology and Oncology, China Medical University Hospital, Taichung, Taiwan. 3Department of Internal Medicine, Several genes related to lung cancer metastatic to the brain China Medical University Hospital, Taichung, Taiwan. 4Center for Molecular that play a role in cancer cells' adhesion to brain endothelial 5 Medicine, China Medical University Hospital, Taichung, Taiwan. Graduate cells include N-cadherin, integrin, and a disintegrin and metal- Institute of Physiology, National Taiwan University, Taipei, Taiwan. 6Yong- Lin Biomedical Engineering Center, National Taiwan University, Taipei, loproteinase domain 9 (ADAM9; refs. 5 and 6). ADAM9 belongs Taiwan. 7Department of Pathology, China Medical University Hospital, to the ADAM family of type I transmembrane proteins, which Taichung, Taiwan. 8Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 9Depart- are involved in several biologic processes, including neurogen- ment of Biotechnology, Asia University, Taichung, Taiwan. 10PhD Program esis, angiogenesis, and cell adhesion and migration (7). The for Translational Medicine, College of Medical Science and Technology, disintegrin domain of ADAM9 adheres to cells by binding to Taipei Medical University, Taipei, Taiwan. 11Graduate Institute for Cancer Biology, China Medical University, Taichung, Taiwan. integrins (8) or heparin sulfate proteoglycans (9), and the metalloproteinase domain functions in ectodomain shedding Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). to release a variety of cell-surface proteins, including growth factors, cytokines, cell adhesion molecules, and receptors (10). Present address of T.-P. Lu: Department of Public Health, National Taiwan Overexpression of ADAM9 has been consistently observed University, Taiwan. in pancreatic, breast, and prostate cancers (11–13). Using an Corresponding Authors: Mien-Chie Hung, Department of Molecular and ﬁ Cellular Oncology, The University of Texas MD Anderson Cancer Center, arti cial overexpression system, investigators showed that 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3668; ectopic expression of ADAM9 in lung cancer cells correlated Fax: 713-794-0209; E-mail: [email protected]; or Yuh-Pyng Sher, with metastases to the brain through the regulation of Graduate Institute of Clinical Medical Science and Center for Molecular Medicine, China Medical University and Hospital, Taichung 404, Taiwan. integrin a3andb1 (6). To further understand the mechan- Phone: 886-4-22052121 ext. 7819; Fax: 886-4-2233-3496; E-mail: isms by which ADAM9 promotes lung cancer metastatic to [email protected] the brain in addition to ADAM9's role in cancer cells' doi: 10.1158/0008-5472.CAN-13-2995 adhesion to brain endothelial cells, we analyzed the differ- 2014 American Association for Cancer Research. ential gene expression between control cells and ADAM9

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knockdown lung cancer cells metastatic to the brain and of the specific gene were higher than the median in all investigated the ADAM9-related pathways required for lung samples; and (ii) a low group, in which gene expression levels cancer metastasis to the brain. We also analyzed tissue of the specificgenewerelowerthanthemedianinall samples from lung adenocarcinoma patients to investigate samples. Kaplan–Meier survival curves were used to assess the clinical relevance of ADAM9. the differences between these two groups. Finally, a Cox hazard regression model was used to evaluate the quanti- Materials and Methods tative risk of the gene expression grouping in patients with Cell lines and animal model lung adenocarcinoma. Human lung adenocarcinoma cell lines CL1-0 (low invasiveness) and F4 (high invasiveness) from the same lung cancer Immunohistochemistry staining origin (14) and F4-luc cells with stable expression of high levels The primary and metastatic specimens were obtained from fi of luciferase were established as previously described (15). the les of the Department of Pathology of China Medical Experiments were carried out within 6 months of acquiring the University Hospital in compliance with protocols approved by n ¼ cells from the established cell bank to ensure that cells main- the CMUH IRB (DMR99-IRB-131). Group I ( 18), primary tained their ability to form lung cancer tumors in SCID mice. lung tumor from patient with early-stage disease without n ¼ All cell lines were tested to be free of mycoplasma and relapse for more than 4 years; group II ( 24), primary lung authenticated by short tandem repeat (STR) DNA typing tumor from patients with early-stage disease that relapsed in 1 n ¼ (Genelabs Life Science) in November 2013. To generate cell to 3 years; group III ( 21), primary lung tumor from patients lines of lung cancer metastatic to the brain, parental F4-luc with late-stage disease metastatic to brain or bone at diagnosis; n ¼ cells (5 104 cells) were injected intracardially into 8-week-old group IV ( 21), paired lung tumor metastatic to brain or SCID mice (n ¼ 5; BioLASCO) and imaged weekly by in vivo bone from patients in group III. Asian lung cancer tissue array imaging system (IVIS) spectrum imaging system (Xenogen) for was purchased from US Biomax (LC1006). ADAM9, CDCP1, 1 month under SPF condition. When brain metastases were and tPA expression in clinical specimens from patients with detected by IVIS, the whole brain was dissected, minced, lung cancer were detected by immunohistochemical staining fl treated with collagenase type IA (0.2 mg/mL; Sigma) for 1 hour, as previously described (15). Brie y, goat anti-mouse ADAM9 and then washed and cultured in DME/F12 plus 10% FBS antibody (AF949; Abcam; ref. 13), goat anti-human CDCP1 media to get clones of the lung cancer cells metastatic to the antibody (AB1377; Abcam), and rabbit anti-human tPA (sc- brain such as Bm2 and Bm7. Bm7 cells were injected intra- 5241; Santa Cruz Biotechnology) were used to perform IHC – – cardially into SCID mice again, and the same process was staining using horseradish peroxidase conjugated avidin bio- repeated to verify the cells' ability to metastasize to the brain. tin complex (ABC) from the Vectastain Elite ABC Kit (Vector Bm7brm cells were obtained from the second cycle (Fig. 1A). Laboratories) and AEC chromogen (Vector Laboratories). The To avoid increasing the risk of genetic mutation during the sections were counterstained with hematoxylin and mounted. prolonged period in which cells were generated, Bm7 cells were All stainings were evaluated by experienced histologists. used as brain-metastatic cells in this study. All animal experi- Results ments were carried out under protocols approved by the Institutional Animal Care and Use Committee of China Med- ADAM9 expression is associated with lung cancer ical University and Hospital. metastasis to the brain In a series of established human lung cancer cell sublines metastatic to the brain (see Materials and Methods; left, Fig. Microarray processing and survival analyses 1A), ADAM9 protein expression in sublines Bm7 and Bm7brm Three public microarray datasets were analyzed: two were was higher than ADAM9 expression in the low metastatic downloaded from the Gene Expression Omnibus (16), includ- CL1-0 and high metastatic F4 cells (Fig. 1A, right). The Bm7 ing GSE8894 (61 patients in South Korea, two patients with cells showed stronger metastatic ability in vitro and were more adenocarcinoma were excluded because age was unavailable; drug resistant than the F4-luc parental cells were (Supplemen- ref. 17) and GSE11969 (90 patients in Japan; ref. 18), and the tary Fig. S1A and S1B), a finding consistent with clinical third dataset, composed entirely of lung adenocarcinoma observations in patients with lung cancer with brain metas- patients, was obtained from the Shedden and colleagues study tases (22). Furthermore, Bm7 cells maintained the luciferase (469 patients in the United States; ref. 19). For our analysis, only activity of the original F4-luc cells for establishing an animal patients with lung adenocarcinoma from each of the datasets model of lung cancer metastatic to the brain (Supplementary were examined. We expanded the number of patient for the Fig. S1C). survival curve analysis by combining the GSE8894 and We generated two Bm7 ADAM9 knockdown cell lines (shA- GSE11969 datasets (referred to as Asia datasets). After retriev- DAM9-C and shADAM9-E) by lentivirus expressing shRNAs ing the raw data, a quantile normalization method was per- targeting different regions of ADAM9. The expression of formed for each dataset to remove the potential systematic ADAM9 was more strongly inhibited in shADAM9-E than in biases. Survival analyses were performed based on the proce- shADAM9-C cells (Fig. 1B, top). Bm7 cells with ADAM9 knock- dures in our previous studies (20, 21). Briefly, for each gene, down (Bm7-shADAM9-C and Bm7-shADAM9-E) had a signif- samples were divided into two groups according to the fol- icantly weaker migration rate than shGFP control cells had lowing criteria: (i) a high group, in which gene expression levels (Fig. 1B, bottom) but had a similar proliferation rate to controls

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ADAM9 Promotes Lung Cancer Metastases to Brain

Figure 1. Knockdown of ADAM9 decreases lung cancer metastatic to the brain. A, ﬂowchart for establishing cell sublines of lung cancer metastatic to the brain. F4-luc cells were injected intracardially into SCID mice and cultured from dissected brain tissue. The cycle was repeated twice to establish the brain- metastatic sublines Bm7 and Bm7brm. Western blot analysis of ADAM9 expression is shown. ADAM9 proteins were detected in nonreducing gels using the antibody from R&D (MAB939; R&D Systems). B, migration ability of ADAM9 knockdown (Bm7-shADAM9) and control (Bm7-shGFP) cells was evaluated by wound-healing assay. Western blot analysis of ADAM9 protein expression is shown at the top. EF1a served as protein-loading control. C, trans-BBB migration assay of ADAM9 knockdown and control cells in vitro. Top, the images of trans-migrated cells. Bottom, quantitation of the number of trans-migrated cells from three independent experiments. , P < 0.05 and , P < 0.01. D, relative binding activity of each indicated cell line to HBMEC at 0.5 and 2 hours. The binding activity of Bm7-shGFP at 2 hours was set as 100% for comparison between cell lines. , P < 0.01. E, Bm7-shGFP, Bm7-shADAM9-C, or Bm7-shADAM9-E cells were injected intracardially into SCID mice and monitored under IVIS detection. The number of mice in which the result was detected out of the total mice in each group is shown in parenthesis. F, the metastatic lung tumor from the brain of mice bearing Bm7-shGFP cells was conﬁrmed by pathological hematoxylin and eosin staining.

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within 2 days (Supplementary Fig. S1D). Results from the in noticed that ADAM9 and CUB-domain–containing protein 1 vitro blood–brain barrier assay indicated that there were more (CDCP1) were shown in one interaction network involved in trans-migrated cells in Bm7 than in CL1-0 or F4 cells (Supple- cellular movement (Fig. 2B). The protein levels of ADAM9 and mentary Fig. S1E) but the increase in trans-migrated cells was total CDCP1 (both full-length and cleavage) were increased in attenuated in Bm7 ADAM9 knockdown cells (Fig. 1C). How- cells from low-metastatic (CL1-0) to high-metastatic cell lines ever, adhesion of control and ADAM9 knockdown Bm7 cells to (Fig. 2C). Notably, the cleaved CDCP1 was higher in the brain human brain endothelial cells (HBMEC) was not altered (Fig. metastatic cells (Bm7) than in the F4 cells. We observed similar 1D). Although these data indicated that adhesion to HBMEC results (higher cleaved CDCP1) in the brain metastatic cell required more than ADAM9 alone, all lung cancer cells met- subline MDA-MB-231-brm (24) than in the parental MDA-MB- astatic to the brain demonstrated stronger adhesion than 231 breast cancer cell line (Fig. 2C). To further determine parental F4-luc did, further validating that lung cancer cells whether the metalloproteinase activity of ADAM9 proteins metastatic to the brain have a high ability to adhere to brain affects the expression level of CDCP1, we treated lung cancer endothelial cells. Bm7 cells with a broad-spectrum metalloproteinase inhibitor To study the metastatic effect of ADAM9 in vivo, control and BB-94. ADAM9 expression was suppressed under BB-94 treat- Bm7-shADAM9 cells were introduced by intracardiac injection ment (Fig. 2D). In addition, the levels of full-length CDCP1 into SCID mice and monitored for brain metastasis, as this proteins were strongly decreased at 160 mmol/L BB-94 treat- approach has been previously established for a preclinical ment whereas the levels of cleaved CDCP1 were slightly metastasis model for studying metastasis to brain or bone decreased (Fig. 2D). CDCP1 expression was slightly decreased (23). Twenty days after injection, mice that received Bm7- in A549 cells treated with BB-94 (Fig. 2E). These results suggest shADAM9 cells had lower ability of lung cancer cells to that the metalloproteinase activity of ADAM9 mainly affects metastasize to brain and/or bone than did mice in the control the expression of full-length CDCP1 but not of CDCP1 cleavage (Bm7-shGFP) group, as indicated by the loss of luciferase as the latter may be regulated by other factors. signals under IVIS imaging. In the Bm7-shGFP group, all mice CDCP1 is a transmembrane protein involved in cell–cell developed tumor metastasis to brain, bone, and lung whereas interaction and has been shown to function as a regulator of metastasis developed in 80% and 25% of mice in the Bm7- anoikis resistance (which bypasses apoptosis induced by shADAM9-C and Bm7-shADAM9-E groups, respectively (Fig. anchorage-dependent cells detaching from the surrounding 1E). Furthermore, histologic examination of brain sections extracellular matrix) in lung adenocarcinomas (25). CDCP1 is confirmed tumor formation in the brains of mice in the overexpressed in leukemia, metastatic colon cancer, and breast Bm7-shGFP group (Fig. 1F). Taken together, these data indi- cancer (26). De novo expression of CDCP1 in HeLa cells cate that cells with higher levels of ADAM9 are also more dramatically increased their potential to colonize lungs, brain, metastatic and that depletion of ADAM9 significantly reduces and ovaries in vivo (27). However, the molecules that regulate cancer metastasis. CDCP1 expression and activation have not yet been fully identified. To further demonstrate that ADAM9 regulates ADAM9 enhances CDCP1 expression and activation CDCP1 expression, 293 cells with high transfection efficiency Although ectopic expression of ADAM9 in lung cancer cells and low ADAM9 expression were used to overexpress ADAM9. was reported to correlate with brain metastasis through upre- Ectopic expression of ADAM9 in 293 cells increased the full- gulation of integrin a3 and b1 (6), Bm7-shADAM9 cells did not length CDCP1 but not the cleaved form; transient transfection significantly suppress surface integrin b1 compared with sup- of ADAM9 in Bm7 cells increased the full-length and cleaved pression in parental or control cells when we detected total, CDCP1 proteins (Fig. 3A), suggesting ADAM9 increases the active, and inactive integrin b1, except for CL1-0 cells, which level of full-length CDCP1 but does not directly affect the level have low integrin b1 expression (Supplementary Fig. S2). These of CDCP1 cleavage. The full-length CDCP1 ranges from 130 to findings support our previous results showing that Bm7-shA- 140 kDa, which is larger than calculated size of 90 kDa because DAM9 cells did not affect cell adhesion (Fig. 1D). To investigate of extensive glycosylation and is cleaved by a serine protease, how ADAM9 affects lung cancer metastatic to the brain and to for example, matriptase, plasmin, or trypsin (28, 29), through determine which genes are regulated by ADAM9 to promote an unclear mechanism to generate a 70-kDa C-terminal frag- metastasis, we analyzed differential gene transcription profile ment. Proteolysis of CDCP1 induces tyrosine phosphorylation by comparing the control cells and ADAM9 knockdown cells in of its C-terminal 70-kDa fragment and recruitment of Src and 2 cell sublines of lung cancer metastatic to brain, Bm2 and PKCd to initiate signaling pathways (30). In Bm7-shGFP cells, 2 Bm7, as well as comparing Bm2 and Bm7 with parental F4-luc forms of CDCP1 proteins were detected, and the levels of the cells. Genes with more than 2-fold change were considered as cleaved form were lower in Bm7-shADAM9-E cells (Fig. 3B). likely to be involved in the ADAM9-associated lung cancer The levels of tyrosine-phosphorylated-CDCP1-interacting sig- metastatic to the brain. A total of 182 differentially expressed naling molecules (phospho-Src and phospho-PKCd) were also genes identified by comparing control with ADAM9 knock- decreased in Bm7-shADAM9-E cells but only slightly affected down and brain-metastatic cell sublines with parental cells in Bm7-shADAM9-C cells; that difference was associated with (Supplementary Table S1) was used in hierarchical clustering ADAM9 expression (Fig. 3B). To further determine whether analysis (Fig. 2A). Using the Ingenuity Pathway Analysis net- ADAM9-enhanced CDCP1-interacting signaling molecules was work generation algorithm to determine highly interconnected because of the more stable cleaved CDCP1 rather than to the pathways representing fundamental biologic functions, we full-length CDCP1, the degradation rate of two forms of CDCP1

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ADAM9 Promotes Lung Cancer Metastases to Brain

Figure 2. ADAM9-regulated genes and associated networks in lung cancer cells metastatic to brain identiﬁed from transcriptome microarray analysis. A, hierarchical clustering of the 182 differentially expressed genes by comparing control versus ADAM9 knockdown and brain-metastatic cell sublines (Bm2 and Bm7) versus parental lung cancer cells. Red, genes that were upregulated; green, downregulated genes. B, Ingenuity Pathway Analysis showing the gene network in cellular movement, molecular transport, and neurologic disease. C, Western blot analysis of ADAM9 and CDCP1 in parental cells and brain-metastatic cell sublines (left, lung cancer cells; right, breast cancer cells). The numbers below the middle panel represent the levels of full-length and cleaved CDCP1 in F4 and Bm7 cells relative to CL1-0 cells. D, Western blot analysis of ADAM9 and CDCP1 in Bm7 cells treated with BB-94, a broad-spectrum inhibitor of metalloproteinases. The numbers below the top panel represent the levels of ADAM9 expression in BB-94–treated relative to DMSO-treated Bm7 cells. The numbers below the middle panel represent the levels of full-length and cleaved CDCP1 in BB-94–treated relative to DMSO-treated Bm7 cells. E, Western blot analysis of ADAM9 and CDCP1 in A549 cells treated with BB-94. The numbers below the top panel represent the levels of ADAM9 expression in BB-94–treated relative to DMSO-treated A549 cells. The numbers below the middle panel represent the levels of full-length and cleaved CDCP1 in BB-94–treated relative to DMSO-treated A549 cells. Western blots shown in C–E were probed with anti-ADAM9 antibody (#2099; Cell Signaling Technology) under reducing conditions. EF1a served as protein-loading control. was determined. Under cycloheximide treatment to block Both forms of CDCP1 were tyrosine phosphorylated, an protein neosynthesis, the levels of full-length CDCP1 decreased event required to overcome anoikis (25), and no difference after 6 hours whereas the levels of cleaved CDCP1 remained between the phospho-CDCP1 signal and total CDCP1 proteins high for 24 hours in control cells. The calculated degradation in control or shADAM9-E cells was observed, suggesting that rate of full-length CDCP1 was 2.9% per hour whereas almost no both forms (full-length and cleavage) of CDCP1 are function- degradation of the cleaved form was observed, indicating that ally active (Fig. 3D). To determine whether suppression of cleaved CDCP1 is more stable than the full-length form (Fig. CDCP1 cleavage and CDCP1-interacting signaling molecules in 3C). Thus, ADAM9 likely enhances the CDCP1 signaling as ADAM9 knockdown cells inﬂuences the function of CDCP1 in more cleaved CDCP1 is present in the control cells. anoikis resistance, we performed a soft agar assay and found

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Figure 3. ADAM9 enhances cleaved CDCP1 protein level and the function of CDCP1 in anoikis resistance. A, Western blot analysis of ADAM9 and CDCP1 in 293 cells transfected with ADAM9 expression or control vector. B, Western blot analysis of expression of CDCP1 and its downstream proteins in control and ADAM9 knockdown cells. P/T, the quantiﬁed ratio of phosphorylated proteins to total proteins. ADAM9 proteins were detected in nonreducing gels using the antibody from R&D (MAB939; R&D Systems). C, protein stability of ADAM9 and CDCP1 in ADAM9 knockdown cells treated with cycloheximide at different incubation times was analyzed by Western blot (top) and quantiﬁed (bottom). The intensity of the band at 0 hour was used as a control (100%). D, detection of CDCP1 phosphotyrosine levels. Total CDCP1 proteins were immunoprecipitated from control and ADAM9 knockdown cells and detected using anti-phosphotyrosine antibody. E, colony formation of ADAM9 knockdown cells by soft agar assay. Representative results showing the number of colonies (each one > 200 mm in diameter) from stained soft agar plates after 4 weeks' growth. (Continued on the following page.)

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that ADAM9 knockdown cells formed lower number of colo- the ratio of cleaved CDCP1 over the full-length CDCP1 and nies than did the parental or control cells (Fig. 3E). In addition, found that CDCP1 cleavage was attenuated in tPA knockdown the number of apoptotic cells was higher in the Bm7-shA- but increased in PAI-1 knockdown cells compared with control DAM9-E than in the control group in an anchorage-free culture cells (Supplementary Fig. S4C). The activity of secreted tPA in condition (Fig. 3F, top and Supplementary Fig. S3A). the cultured media of these cells for plasminogen to plasmin To further investigate the function of the ADAM9–CDCP1 conversion was measured (Fig. 4C and Supplementary Fig. axis, we knocked down CDCP1 in Bm7 cells and found that the S4D), and further analysis showed a high correlation between levels of CDCP1-interacting signaling molecules were attenu- tPA activity and the ratio of CDCP1 cleavage to full-length ated but not those of ADAM9 (Supplementary Fig. S3B). A CDCP1 (R ¼ 0.89) among these cancer cells (Fig. 4D). The similar trend of increasing number of apoptotic cells in an percentage of apoptotic cells in an anchorage-free culture anchorage-free culture condition was observed in CDCP1 condition was increased in tPA knockdown cells over the knockdown cells (Fig. 3F, bottom, and Supplementary Fig. percentage in the control cells (Fig. 4E and Supplementary S3A and S3C), consistent with the known function of CDCP1 Fig. S3A), which is consistent with our observation in ADAM9 in anoikis resistance. Ectopic expression of CDCP1 in Bm7- and CDCP1 knockdown cells and indicates that tPA plays a role shADAM9-E cells rescued the cells' migration ability to a level in anoikis resistance. comparable to that of Bm7-shGFP (Fig. 3G). Thus, ADAM9 To test whether tPA addition could enhance CDCP1 cleav- increases CDCP1 expression and the function of CDCP1 in age, Bm7-shADAM9-E cells were treated with recombinant tPA anoikis resistance and metastasis. proteins. In 10% serum culture media, cleaved CDCP1 was slightly increased at a higher dose of tPA treatment. Interest- ADAM9 enhances CDCP1 cleavage through tPA ingly, CDCP1 cleavage in Bm7-shADAM9-E cells treated with activation 100 nmol/L tPA was also enhanced to a level comparable with Several proteases, including matriptase and plasmin, have that in the control under serum-free conditions (Fig. 4F); this been shown to cleave CDCP1. Tissue plasminogen activator finding indicated that CDCP1 is likely a substrate of tPA. (tPA), a serine protease responsible for the degradation of Next, to investigate whether tPA directly cleaves CDCP1, blood clots by cleaving plasminogen to become plasmin, is full-length CDCP1 was isolated and treated with tPA and then necessary and sufficient to induce opening of the BBB that analyzed by Western blotting. Plasmin and trypsin, which are causes BBB breakdown in a plasmin-dependent or plasmin- known to cleave CDCP1, were included as the control. The independent manner (31, 32). No matriptase protein in Bm7 conversion of full-length to cleaved CDCP1 was increased in a cells (Supplementary Fig. S4A) and a near background level of dose-dependent manner with tPA treatment similar to plas- plasminogen RNA from transcriptome analysis in lung cancer min and trypsin treatment, indicating that tPA can directly cells were detected. Notably, from the transcriptome analysis cleave CDCP1, albeit at a high concentration (Fig. 4G). These list (Supplementary Table S1), high tPA (PLAT) expression and results suggest that tPA activation mediates ADAM9-enhanced low plasminogen activator inhibitor 1 (PAI-1, SERPINE1) CDCP1 cleavage. expression were observed in cancer cells metastatic to the brain (Fig. 2A). We further investigated whether ADAM9- Blocking the ADAM9–CDCP1–tPA axis in lung cancer regulated CDCP1 cleavage requires tPA activation by examin- cells prolongs animal survival time ing both tPA and PAI-1 expression. Because of the ADAM9 Next, we detected the migration rate of Bm7 cells by levels in Bm7-shADAM9-C and -E remained high in the pooled silencing genes at the ADAM9–CDCP1–tPA axis. We showed population, we selected the stable clones with low ADAM9 that control cells migrated farther and in a wider range of expression for further experiments. The reduced levels of direction than Bm7-shADAM9, Bm7-shCDCP1, and Bm7- ADAM9 level in ADAM9 knockdown cells substantially shtPA cells did, whereas Bm7-shPAI-1 rescued the migration decreased expression of full-length and cleaved CDCP1 (Fig. ability (Fig. 5A, top). Quantitation of cellular movement con- 4B). In addition, the mRNA and protein levels were lower for firmed that the loss of gene expression at ADAM9–CDCP1–tPA tPA and higher for PAI-1 in ADAM9 knockdown stable clones axis significantly inhibited cell migration (Fig. 5A, bottom). than in the control cells (Fig. 4A and B). The catalytic activity of These cells were further investigated for their metastatic ability tPA for plasminogen to plasmin conversion was also sup- in animal models by intracardiac injection in mice. Kaplan– pressed in ADAM9 knockdown cells (Fig. 4C). Meier analysis indicated that mice inoculated with Bm7-shA- To investigate whether the activity of tPA correlates with DAM9 and Bm7-shtPA cells had a significantly longer overall ADAM9-regulated CDCP1 cleavage, tPA and PAI-1 knockdown survival than did mice inoculated with the control cells. cells (Supplementary Fig. S4B) were used to investigate the Notably, mice in the Bm7-shCDCP1 group had only moderately correlation of CDCP1 cleavage with tPA activity. We measured prolonged survival, but mice in the Bm7-shtPA group had the

(Continued.) F, the percentage of apoptosis in Bm7-shGFP, Bm7-shADAM9E, Bm7-shCDCP1-A1, and Bm7-shCDCP1-B2 cells was determined by ﬂow cytometry using Annexin-V (high) and PI staining (low) under anchorage-free culture conditions at different time points. Each experiment was repeated independently and showed a similar trend. Raw data are shown in Supplementary Fig. S3A. G, plasmid mixture of CDCP1 and GFP (molar ratios ¼ 5:1) was transiently transfected into Bm7-shADAM9-E cells. After 24 hours, the 16-hour migration distance (motile activity) of Bm7-shADAM9-E cells was measured by time-lapse video microscopy in each group (left) and quantiﬁed (right). Transfected Bm7-shADAM9-E cells with high GFP signal represent CDCP1-transfected group, whereas low GFP signal represents control cells. Error bars, SD; , P < 0.01. Western blot analysis of CDCP1 is shown at the top.

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Figure 4. The tPA activity correlates with conversion of full-length CDCP1 to the cleavage form. A, RT-qPCR of tPA (top) and PAI-1 (bottom) in control and ADAM9 knockdown cells. B, Western blot analysis of ADAM9, CDCP1, tPA, and PAI-1 expression in control and ADAM9 knockdown cells. C, activity of tPA in different conditioned media from the indicated cell cultures. , P < 0.01. D, activity of tPA versus the ratio of CDCP1 cleavage over the full-length form; R ¼ 0.89. E, the percentageofapoptoticcellswasdeterminedbyﬂowcytometryunderanchorage-freecultureconditionsatdifferenttimepoints.(Continuedonthefollowingpage.)

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Figure 5. Blocking the ADAM9–CDCP1–tPA axis in lung cancer cells prolongs animal survival time. A, the migration distance (motile activity) for ADAM9, CDCP1, tPA, and PAI-1 knockdown cells was measured by time-lapse video microscopy (top) and quantiﬁed (bottom). Error bars, SD from three independent experiments; , P < 0.05; , P < 0.01. B, Bm7 cells with indicated shRNA knockdown were intracardially injected into SCID mice and mice survival time was monitored for 50 days. The number (n) of mice shown in each group was from two independent experiments. C, time-course analysis of sub-G1 fraction from the cell cycle. Knockdown cells as indicated were cultured in anchorage-free conditions and subjected to cell-cycle analysis. Sub-G1 fraction values at 0, 24, and 48 hours are shown. D, Western blot analysis of indicated proteins in Bm7 cells after dexamethasone (dexa) or dasatinib (dasa) treatment for 24 hours. Media-alone treatment served as negative control. E, cytotoxic effects of dexamethasone and/or dasatinib after 72-hour treatment. Dose- dependent cytotoxic fraction (top); combination index (CI) summary (bottom) at 50%, 75%, and 90% inhibition (ED50, ED75, and ED90) for two cell lines; treatments with a combination of dexamethasone and dasatinib are shown. A CI value <1 indicates synergism.

(Continued.) Raw data are shown in Supplementary Fig. S3A. F, Bm7-shADAM9-E cells were cultured with or without serum and treated with different concentrationsoftPA(BoehringerIngelheim).ThenumbersbelowtheCDCP1blotrepresentthelevelsofCDCP1cleavageincellstreatedwithtPAtreatmentrelative tocellswithouttPAtreatmentinserum-containingor-freeculturecondition.G,WesternblotanalysisofimmunoprecipitatedCDCP1proteinsinADAM9knockdown stable cells treated with indicated proteases (top). The numbers below the panel represent the ratio of CDCP1 cleavage over full-length and were plotted against various concentrations of protease treatment (bottom). Stable clones of ADAM9 knockdown cells were used instead of the pooled population.

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Figure 6. High expression of ADAM9 and CDCP1 in primary tumors predicts mortality in lung adenocarcinoma patients. A, Kaplan–Meier survival analyses of patients with lung adenocarcinoma in the indicated groups calculated by dividing the patients with lung adenocarcinoma into different groups via the gene expression level in three independent datasets. H, high expression; L, low expression above or below the median, respectively. DH, SH, and DL indicate dual- high, single-high, and dual-low expression of ADAM9 and CDCP1, respectively. (Continued on the following page.)

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longest survival time (Fig. 5B). When these cells were cultured icantly higher risk of lung cancer mortality than do patients Low Low in anchorage-free conditions, the sub-G1 percentage, which with ADAM9 /CDCP1 expression in both the Asia measures the apoptotic cell population, was higher in the Bm7- and Western datasets (hazard ratios of 5.97 and 1.80, shtPA, moderately higher in the Bm7-shADAM9, and slightly respectively; Fig. 6B). To rule out statistical bias as the reason higher in the Bm7-shCDCP1 cells than in the control cells in for these findings, we compared the predictability of ADAM9 anchorage-free conditions (Fig. 5C). These results provide a versus CDCP1, and selected the more predictive of these two partial explanation for the longer survival time in the Bm7- genes; the selected gene in random combination with one of shtPA group. other genes was then used to generate a profile that illustrates Next, we examined whether targeting the ADAM9–CDCP1– the predictability for the different pairs of genes. This gene tPA axis could benefit lung cancer treatment using clinically predictability profile indicated that the predictor of ADAM9 available drugs such as dexamethasone and dasatinib. Dexa- plus CDCP1 ranked among the top 3.3% (GSE8894), 7.9% methasone has been reported to inhibit CDCP1 cleavage (GSE11969), and 18.6% (Shedden datasets) of random combi- through upregulation of PAI-1 (33). Dasatinib is a dual Src/ nation (Fig. 6C). A number that is less than 20% indicated that Abl kinase inhibitor that also targets several other kinases (34) our finding in prognosis analysis is not from statistical bias; with potent antiproliferative activity against hematologic thus, patients with lung adenocarcinoma with ADAM9High/ malignancies and cancer cell metastasis (35). We found that CDCP1High expression indeed had a higher risk of mortality dexamethasone enhanced PAI-1 expression in Bm7-shGFP than those with ADAM9Low/CDCP1Low expression. Moreover, cells and enhanced PAI-1 expression even more strongly in Fisher exact test indicated that the percentage of patients with Bm7-shADAM9 cells and that dasatinib significantly inhibited high expression of tPA was significantly higher (67%) in the the levels of pSFK and pPKCd in both cells (Fig. 5D). Moreover, ADAM9High/CDCP1High group than those in the ADAM9Low/ the 2-drug treatment combination acted synergistically in Bm7 CDCP1Low group in the Asia datasets (37%; Fig. 6D) and cells and induced a dose-dependent cytotoxic effect (Fig. 5E, Western (Fig. 6E) datasets (37%; P < 0.05). These results top; CI < 1; Fig. 5E, bottom). These findings suggest that support the clinical relevance of ADAM9High/CDCP1High in blocking the ADAM9–CDCP1–tPA axis inhibits lung cancer patients via its association with poor prognosis and imply that cell growth and metastasis. ADAM9High/CDCP1High expression in tumors is associated with cancer metastasis. Overexpression of ADAM9 and CDCP1 in primary lung adenocarcinoma is associated with a poor prognosis High expression of ADAM9, CDCP1, and tPA in metastatic From above, we demonstrated that ADAM9 enhances lung adenocarcinoma CDCP1 activation through tPA for anchorage-free survival of To assess the clinical relevance of ADAM9 protein expres- cancer cells. This finding raises the question of whether sion in patients with lung cancer, we performed ADAM9 activation of the ADAM9–CDCP1 axis in patients with lung immunohistochemical (IHC) staining in a lung cancer tissue adenocarcinoma has clinical relevance owing to its association array from the Asian patients. ADAM9 expression was high in with poor prognosis. In the Asia datasets, patients with high lung adenocarcinoma tissue but remained low expression in ADAM9 (ADAM9High) expression had significantly shorter matched adjacent normal lung tissue (P ¼ 0.012; Fig. 7A). Most survival times than did patients with low ADAM9 (ADAM9Low) of the lung lesions metastatic to the brain had elevated ADAM9 expression (P ¼ 0.0008). Similar results were observed in mRNA expression compared with expression in bone meta- patients with high CDCP1 (CDCP1High) expression, who also static lesions or normal lung tissues (P ¼ 0.01; Fig. 7B). To had significantly shorter survival times than did those with low evaluate whether activation of the ADAM9–CDCP1 axis con- CDCP1 (CDCP1Low) expression (P ¼ 0.015; Fig. 6A, top). tributes to lung cancer metastasis, we performed IHC staining Conversely, in the Western datasets (Shedden), ADAM9 expres- of 4 groups of clinical paraffin block tissue specimens from sion level in patients did not correlate with patient prognosis lung adenocarcinoma patients at the China Medical University (P ¼ 0.174), whereas patients with CDCP1High expression had Hospital (see Materials and Methods). Comparison of ADAM9 significantly shorter survival times than did those with expression in these specimens showed that the rate of positive CDCP1Low expression (P ¼ 0.0002; Fig. 6A, bottom). However, ADAM9 detection increased almost 2-fold in groups II and III when we combined ADAM9 and CDCP1 as a predictor, patients (54% and 52%) and reached a 3-fold increase in group IV (81%) with ADAM9High/CDCP1High expression had a significantly compared with group I (28%; Fig. 7C). The rate of positive worse prognosis than did patients with ADAM9Low/CDCP1Low CDCP1 detection remained high in all 4 groups (from 67% to in the Asia and Western datasets (Fig. 6A). 71%; Fig. 7C). The rate of positive tPA detection increased from Cox hazard regression model analysis also showed that 22% to 81% (Fig. 7C). The distribution of ADAM9 staining patients with ADAM9High/CDCP1High expression have a signif- significantly correlated with the tPA staining results (P ¼

(Continued.) The number of patients (n) in each group is shown. B, Cox proportional hazard regression analyses of overall survival of patients with lung adenocarcinoma with ADAM9 or CDCP1 expression. C, ranking of the P value of ADAM9 plus CDCP1 among the null P-value baseline obtained by combining a ﬁxed predictor with the other genes randomly selected from the original gene pool. Fixed predictor: ADAM9 in the GSE11969 dataset and CDCP1 in the GSE8894 and Shedden datasets. P value of ADAM9High/CDCP1High versus ADAM9Low/CDCP1Low is marked by the red line. D, comparison of tPA expression in DH, SH, and DL patient groups in combined GSE8894 and GSE11969 datasets using Fisher exact test. E, comparison of tPA expression in DH, SH, and DL patient groups in the Shedden dataset using Fisher exact test.

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Figure 7. Detection of ADAM9, CDCP1, and tPA protein expression in human lung cancer specimens. A, IHC analysis of ADAM9 in lung cancer tissue array scored by staining intensity from 0 to 3þ (0, negative; 1, weak; 2, moderate; 3, strong) by histologists. Matched lung adenocarcinoma tissue and adjacent normal lung tissue from the same patients were analyzed for the distribution of ADAM9 staining by the McNemar method. Representative staining for ADAM9 is shown below the table. B, RT-qPCR analysis of ADAM9 mRNA expression in lung cancer cells and metastatic lung cancer specimens from different patients. HPRT was served as internal control. N, normal part; mets, metastatic tumor. C, IHC analysis of ADAM9/CDCP1/tPA expression in 84 clinical paraffinblock specimens from four groups of lung adenocarcinoma patients. A score 1þ indicates positive detection. D, correlation of ADAM9 and tPA expression in all 84 clinical paraffin block specimens. E, trend-change plots of staining scores in paired primary lung (group III) and metastatic lung tumors (group IV). Each line indicates the trend change between a primary and metastatic lung tumor. Red, increasing trend; blue, decreasing trend; black, no changes in the trend; P value was calculated by Wilcoxon signed ranks test. F, the indicated detection rate of ADAM9/CDCP1/tPA in all four groups as defined in C.

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0.034; Fig. 7D). On comparing the IHC staining scores in paired the brain. In addition, enhanced transmigration of the BBB by tumor specimens (primary and metastatic) from the same cancer cells seems to be a result of the enhanced expression of patient, the trend toward higher ADAM9 expression in met- ADAM9 that triggers the activity of tPA. astatic tumor tissue was significantly increased only in patients The catalytic unit of tPA is known to function autonomously with brain metastasis (P ¼ 0.03) but not in patients with bone (37), and there is no report showing ADAM9 cleaves tPA. metastasis (P ¼ 0.53; Fig. 7E). The trend was absent in CDCP1 Although we did not provide sufficient evidence to demon- or tPA staining in patients with metastasis to brain or bone strate the role of ADAM9 protease activity in regulating the (Fig. 7E). By setting the IHC score 1þ as positive staining, the CDCP1–tPA axis, knocking down ADAM9 not only downre- triple-gene-positive (ADAM9–CDCP1–tPA positive staining) gulated the transcription of tPA but also enhanced transcrip- detection rate was low in patients without metastasis (group tion of PAI-1, which suppressed the catalytic activity of tPA for I) but increased in patients with metastasis (groups II and III) plasminogen conversion to plasmin (Fig. 4C). This suggests and even reached 50% in tumor specimens metastatic to brain that ADAM9 indirectly regulates the tPA activity. or bone (group IV; Fig. 7F). In contrast, the triple-gene-negative The mechanism by which ADAM9 increases the expres- (ADAM9–CDCP1–tPA negative staining) detection rate sion of CDCP1 is still unclear. Heparin-binding EGF-like decreased from 22% in the nonmetastatic group to 5% in the growth factor (HB-EGF), a member of EGF family, is over- metastatic groups and even to zero in patients with tumor expressed and associated with carcinogenesis in hepatoma metastatic to brain or bone. Taken together, these clinical and pancreatic cancer (38, 39). The transmembrane domain results indicate that activation of the ADAM9–CDCP1–tPA of HB-EGF (pro-HB-EGF) can be cleaved by metallopro- axis contributes to lung cancer metastasis, especially to the teases such as ADAM9, yielding a secreted form that acti- brain. vates EGFR (40). Activation of EGFR signaling has been reported to upregulate CDCP1 mRNA and protein expression (41). Interestingly, we also observed increased mRNA Discussion levels of HB-EGF in the brain-metastatic Bm7 cells com- Here, we address the potential implications of ADAM9 in paredwithparentalcells.Therefore,itisplausiblethat lung cancer metastatic to the brain and reveal a novel con- ADAM9 increases the expression of CDCP1 via ADAM9- nection between ADAM9 and CDCP1 by which ADAM9 dependent shedding of HB-EGF, leading to EGFR activation increases the cleavage form of CDCP1 through activation of and subsequent upregulation of CDCP1 protein and mRNA tPA by enhancing tPA and suppressing PAI-1 expression. The expression. In addition, EGF has also been reported to contribution of the ADAM9–CDCP1–tPA axis to lung cancer increase tPA mRNA in HeLa cells (42) and enhance PAI-1 metastasis was validated in the cell model, the animal model transcription via sequential activation of c-Src and PKCd in and clinical samples. On the basis of our results, blocking this glioma cells (43). However, we observed downregulation of axis in cancer cells is expected to provide a therapeutic benefit, tPA and upregulation of PAI-1 in ADAM9 knockdown cells, as knockdown of each one of the genes in the axis reduced suggesting other molecules besides EGF are involved in their cancer cell migration and prolonged survival time in mouse regulation. PAI-1 transcription can be regulated by many tumor models. In addition, targeting tPA activity and tyrosine- transcription factors such as hypoxia-inducible factor-1a phosphorylated-CDCP1-interacting signaling molecules, phos- (HIF-1a) and early growth response protein 1 (EGR1). pho-Src with dexamethasone and dasatinib showed a syner- Although EGR1 is quickly activated under hypoxia, it can gistic cytotoxic effect in lung cancer cells. induce transcription and expression of PAI-1 independently Although several studies have focused on ADAM9 as a new of HIF-1a in murine macrophages (44). From the transcrip- anticancer drug target (36), clinical evidence of ADAM9 expres- tome analysis (Supplementary Table S1), we notice that sion in lung cancer is still lacking (6). Our analysis of 3 lung EGR1 mRNA levels were 10-fold higher in ADAM9 knock- cancer datasets and clinical samples suggested that ADAM9 down cells than in control cells, suggesting that ADAM9 may alone is a good prognostic marker in the Asia datasets but not influence expression of PAI-1 through activation of EGR1, in the U.S. datasets. However, the combination of ADAM9 and although more studies will be required to validate this. CDCP1 appears to be a better prognostic marker for patient ThebiologicbehaviorandfunctionofPAI-1seemspar- outcome, implying these genes play important roles in cancer adoxical. PAI-1 is thought to inhibit activation of tPA or uPA, metastasis. Such clinical evidence and results of our study do an event that correlates with poor clinical outcomes in not indicate that ADAM9 is related specifically to metastases to cancer (45, 46); however, PAI-1 also serves as an indicator the brain. Rather, they support that ADAM9 enhances the of poor prognosis in breast cancer (47). These findings may ability of cancer cells to establish distant metastases such as to be attributed to differences in cell types and experimental the brain. By investigating the paired clinical tumor specimens models. Our data indicate that PAI-1 knockdown increases from the tumor's primary site and distant site, albeit in limited tPA activity, which results in more CDCP1 cleavage and cell samples, we found that ADAM9 protein expression was sig- migration ability, implying that PAI-1 inhibits lung cancer nificantly higher in lung tumor tissue metastatic to the brain metastasis. More studies are warranted to clarify the role of than in the primary lung tumor but was not higher in lung PAI-1 in metastasis. tumor tissue metastatic to bone. Although high levels of Here, our findings indicate that ADAM9 regulates a com- ADAM9 in cancer cells are essential for metastasis to the plicated network in lung cancer metastatic to the brain brain, these levels may not be specific to or only limited to through tPA-mediated CDCP1 cleavage, especially in Asian

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patients, and establish a potentially important association Acknowledgments for predicting or preventing cancer metastasis to the brain. The authors thank Diane Hackett from The Department of Scientific Pub- lications at MD Anderson Cancer Center for editing the article. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Grant Support Authors' Contributions This work was supported by grants from National Science Council (NSC99- Conception and design: C.-Y. Lin, H.-J. Chen, S.-Y. Sung, M.-C. Hung, Y.-P. Sher 2314-B-039-029-MY3, NSC101-2325-B-039-008, and NSC102-2628-B-039-010- Development of methodology: S.-Y. Sung, M.-C. Hung, Y.-P. Sher MY3 to Y.-P. Sher; NSC99-2632-B-039-001-MY3 to M.-C. Hung), International Acquisition of data (provided animals, acquired and managed patients, Research-Intensive Centers of Excellence in Taiwan (NSC102-2911-I-002-303 to provided facilities, etc.): C.-Y. Lin, H.-J. Chen, G.-C. Tseng, T.-T. Kuo, M.-C. Hung), Taiwan Department of Health, China Medical University Hospital M.-C. Hung Cancer Research Center of Excellence (CRC; DOH102-TD-C-111-005), CMU102- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, BC-5, and CMU99-NTU-08. computational analysis): C.-Y. Lin, L.-C. Lai, T.-P. Lu, G.-C. Tseng, Q.-Y. Kuok, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked M.-C. Hung advertisement Writing, review, and/or revision of the manuscript: C.-Y. Lin, J.L. Hsu, in accordance with 18 U.S.C. Section 1734 solely to indicate this M.-C. Hung, Y.-P. Sher fact. Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): C.-C. Huang, Q.-Y. Kuok, M.-C. Hung Received October 22, 2013; revised May 19, 2014; accepted June 12, 2014; Study supervision: S.-Y. Sung, M.-C. Hung, Y.-P. Sher published OnlineFirst July 24, 2014.

References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer 16. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI J Clin 2013;63:11–30. gene expression and hybridization array data repository. Nucleic Acids 2. Gavrilovic IT, Posner JB. Brain metastases: epidemiology and path- Res 2002;30:207–10. ophysiology. J Neurooncol 2005;75:5–14. 17. Lee ES, Son DS, Kim SH, Lee J, Jo J, Han J, et al. Prediction of 3. Schwer AL, Gaspar LE. Update in the treatment of brain metastases recurrence-free survival in postoperative non-small cell lung cancer from lung cancer. Clin Lung Cancer 2006;8:180–6. patients by using an integrated model of clinical information and gene 4. Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner expression. Clin Cancer Res 2008;14:7397–404. R, Herms J, et al. Real-time imaging reveals the single steps of brain 18. Matsuyama Y, Suzuki M, Arima C, Huang QM, Tomida S, Takeuchi T, metastasis formation. Nat Med 2010;16:116–22. et al. Proteasomal non-catalytic subunit PSMD2 as a potential ther- 5. Grinberg-Rashi H, Ofek E, Perelman M, Skarda J, Yaron P, Hajduch M, apeutic target in association with various clinicopathologic features in et al. The expression of three genes in primary non-small cell lung lung adenocarcinomas. Mol Carcinog 2011;50:301–9. cancer is associated with metastatic spread to the brain. Clin Cancer 19. Director's Challenge Consortium for the Molecular Classification of Res 2009;15:1755–61. Lung A,Shedden K, Taylor JM, Enkemann SA, Tsao MS, Yeatman 6. Shintani Y, Higashiyama S, Ohta M, Hirabayashi H, Yamamoto S, TJ, et al. Gene expression-based survival prediction in lung ade- Yoshimasu T, et al. Overexpression of ADAM9 in non-small cell lung nocarcinoma: a multi-site, blinded validation study. Nat Med 2008; cancer correlates with brain metastasis. Cancer Res 2004;64:4190–6. 14:822–7. 7. Wolfsberg TG, Primakoff P, Myles DG, White JM. ADAM, a novel family 20. Lu TP, Lai LC, Tsai MH, Chen PC, Hsu CP, Lee JM, et al. Integrated of membrane proteins containing a disintegrin and metalloprotease analyses of copy number variations and gene expression in lung domain: multipotential functions in cell-cell and cell-matrix interac- adenocarcinoma. PLoS ONE 2011;6:e24829. tions. J Cell Biol 1995;131:275–8. 21. Lu TP, Tsai MH, Lee JM, Hsu CP, Chen PC, Lin CW, et al. Identification 8. Nath D, Slocombe PM, Webster A, Stephens PE, Docherty AJ, Murphy of a novel biomarker, SEMA5A, for non-small cell lung carcinoma in G. Meltrin g (ADAM-9) mediates cellular adhesion through a(6)b(1) nonsmoking women. Cancer Epidemiol Biomarkers Prev 2010;19: integrin, leading to a marked induction of fibroblast cell motility. J Cell 2590–7. Sci 2000;113(Pt 12):2319–28. 22. Mehta MP, Paleologos NA, Mikkelsen T, Robinson PD, Ammirati M, 9. Iba K, Albrechtsen R, Gilpin BJ, Loechel F, Wewer UM. Cysteine-rich Andrews DW, et al. The role of chemotherapy in the management of domain of human ADAM 12 (meltrin a) supports tumor cell adhesion. newly diagnosed brain metastases: a systematic review and evidence- Am J Pathol 1999;154:1489–501. based clinical practice guideline. J Neurooncol 2010;96:71–83. 10. Guaiquil V, Swendeman S, Yoshida T, Chavala S, Campochiaro PA, 23. Cruz-Munoz W, Kerbel RS. Preclinical approaches to study the biology Blobel CP. ADAM9 is involved in pathological retinal neovasculariza- and treatment of brain metastases. Semin Cancer Biol 2011;21: tion. Mol Cell Biol 2009;29:2694–703. 123–30. 11. Grutzmann R, Luttges J, Sipos B, Ammerpohl O, Dobrowolski F, 24. Palmieri D, Bronder JL, Herring JM, Yoneda T, Weil RJ, Stark AM, et al. Alldinger I, et al. ADAM9 expression in pancreatic cancer is associated Her-2 overexpression increases the metastatic outgrowth of breast with tumour type and is a prognostic factor in ductal adenocarcinoma. cancer cells in the brain. Cancer Res 2007;67:4190–8. Br J Cancer 2004;90:1053–8. 25. Uekita T, Jia L, Narisawa-Saito M, Yokota J, Kiyono T, Sakai R. CUB 12. O'Shea C, McKie N, Buggy Y, Duggan C, Hill AD, McDermott E, et al. domain-containing protein 1 is a novel regulator of anoikis resistance in Expression of ADAM-9 mRNA and protein in human breast cancer. Int lung adenocarcinoma. Mol Cell Biol 2007;27:7649–60. J Cancer 2003;105:754–61. 26. Hooper JD, Zijlstra A, Aimes RT, Liang H, Claassen GF, Tarin D, et al. 13. Sung SY, Kubo H, Shigemura K, Arnold RS, Logani S, Wang R, et al. Subtractive immunization using highly metastatic human tumor cells Oxidative stress induces ADAM9 protein expression in human prostate identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated cancer cells. Cancer Res 2006;66:9519–26. glycoprotein antigen. Oncogene 2003;22:1783–94. 14. Chu YW, Yang PC, Yang SC, Shyu YC, Hendrix MJ, Wu R, et al. 27. Deryugina EI, Conn EM, Wortmann A, Partridge JJ, Kupriyanova TA, Selection of invasive and metastatic subpopulations from a human Ardi VC, et al. Functional role of cell surface CUB domain-containing lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 1997;17: protein 1 in tumor cell dissemination. Mol Cancer Res 2009;7: 353–60. 1197–211. 15. Sher YP, Tzeng TF, Kan SF, Hsu J, Xie X, Han Z, et al. Cancer targeted 28. Bhatt AS, Erdjument-Bromage H, Tempst P, Craik CS, Moasser MM. gene therapy of BikDD inhibits orthotopic lung cancer growth and Adhesion signaling by a novel mitotic substrate of src kinases. Onco- improves long-term survival. Oncogene 2009;28:3286–95. gene 2005;24:5333–43.

5242 Cancer Res; 74(18) September 15, 2014 Cancer Research

ADAM9 Promotes Lung Cancer Metastases to Brain

29. Casar B, He Y, Iconomou M, Hooper JD, Quigley JP, Deryugina EI. 38. Ito Y, Takeda T, Higashiyama S, Sakon M, Wakasa KI, Tsujimoto M, Blocking of CDCP1 cleavage in vivo prevents Akt-dependent survival et al. Expression of heparin binding epidermal growth factor-like and inhibits metastatic colonization through PARP1-mediated apo- growth factor in hepatocellular carcinoma: an immunohistochemical ptosis of cancer cells. Oncogene 2012;31:3924–38. study. Oncol Rep 2001;8:903–7. 30. He Y, Wortmann A, Burke LJ, Reid JC, Adams MN, Abdul-Jabbar I, 39. Ray KC, Moss ME, Franklin JL, Weaver CJ, Higginbotham J, Song Y, et al. Proteolysis-induced N-terminal ectodomain shedding of the et al. Heparin-binding epidermal growth factor-like growth factor integral membrane glycoprotein CUB domain-containing protein 1 eliminates constraints on activated Kras to promote rapid onset of (CDCP1) is accompanied by tyrosine phosphorylation of its C-terminal pancreatic neoplasia. Oncogene 2014;33:823–31. domain and recruitment of Src and PKCd. J Biol Chem 2010;285: 40. Fischer OM, Hart S, Gschwind A, Prenzel N, Ullrich A. Oxidative and 26162–73. osmotic stress signaling in tumor cells is mediated by ADAM proteases 31. Niego B, Freeman R, Puschmann TB, Turnley AM, Medcalf RL. t-PA- and heparin-binding epidermal growth factor. Mol Cell Biol 2004;24: speciﬁc modulation of a human blood-brain barrier model involves 5172–83. plasmin-mediated activation of the Rho kinase pathway in astrocytes. 41. Dong Y, He Y, de Boer L, Stack MS, Lumley JW, Clements JA, et al. The Blood 2012;119:4752–61. cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) 32. Yepes M, Sandkvist M, Moore EG, Bugge TH, Strickland DK, Lawrence contributes to epidermal growth factor receptor-mediated cell migra- DA. Tissue-type plasminogen activator induces opening of the blood- tion. J Biol Chem 2012;287:9792–803. brain barrier via the LDL receptor-related protein. J Clin Invest 42. Medcalf RL, Schleuning WD. Regulation of human tissue-type plas- 2003;112:1533–40. minogen activator gene transcription by epidermal growth factor and 33. Law ME, Corsino PE, Jahn SC, Davis BJ, Chen S, Patel B, et al. 30,50-cyclic adenosine monophosphate. Mol Endocrinol 1991;5: Glucocorticoids and histone deacetylase inhibitors cooperate to block 1773–9. the invasiveness of basal-like breast cancer cells through novel 43. Paugh BS, Paugh SW, Bryan L, Kapitonov D, Wilczynska KM, Gopalan mechanisms. Oncogene 2013;32:1316–29. SM, et al. EGF regulates plasminogen activator inhibitor-1 (PAI-1) by a 34. LombardoLJ,LeeFY,ChenP,NorrisD,BarrishJC,BehniaK,etal. pathway involving c-Src, PKCd, and sphingosine kinase 1 in glioblas- Discovery of N-(2-chloro-6-methyl-phenyl)-2-(6-(4-(2-hydro- toma cells. FASEB J 2008;22:455–65. xyethyl)- piperazin-1-yl)-2-methylpyrimidin-4- ylamino)thiazole-5- 44. Liao H, Hyman MC, Lawrence DA, Pinsky DJ. Molecular regulation carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with of the PAI-1 gene by hypoxia: contributions of Egr-1, HIF-1a,and potent antitumor activity in preclinical assays. J Med Chem C/EBPa. FASEB J 2007;21:935–49. 2004;47:6658–61. 45. de Witte JH, Sweep CG, Klijn JG, Grebenschikov N, Peters HA, Look 35. Buettner R, Mesa T, Vultur A, Lee F, Jove R. Inhibition of Src family MP, et al. Prognostic value of tissue-type plasminogen activator (tPA) kinases with dasatinib blocks migration and invasion of human mel- and its complex with the type-1 inhibitor (PAI-1) in breast cancer. Br J anoma cells. Mol Cancer Res 2008;6:1766–74. Cancer 1999;80:286–94. 36. Saftig P, Reiss K. The "A Disintegrin And Metalloproteases" ADAM10 46. Borgfeldt C, Bendahl PO, Ferno M, Casslen B. High preoperative and ADAM17: novel drug targets with therapeutic potential? Eur J Cell plasma concentration of tissue plasminogen activator (tPA) is an Biol 2011;90:527–35. independent marker for shorter overall survival in patients with ovarian 37. Kalyan NK, Lee SG, Wilhelm J, Fu KP, Hum WT, Rappaport R, et al. cancer. Gynecol Oncol 2003;91:112–7. Structure-function analysis with tissue-type plasminogen activator. 47. Janicke F, Schmitt M, Graeff H. Clinical relevance of the urokinase-type Effect of deletion of NH2-terminal domains on its biochemical and and tissue-type plasminogen activators and of their type 1 inhibitor in biological properties. J Biol Chem 1988;263:3971–8. breast cancer. Semin Thromb Hemost 1991;17:303–12.

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ADAM9 Promotes Lung Cancer Metastases to Brain by a Plasminogen Activator-Based Pathway

Chen-Yuan Lin, Hung-Jen Chen, Cheng-Chung Huang, et al.

Cancer Res 2014;74:5229-5243. Published OnlineFirst July 24, 2014.

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