Extracellular Protease ADAMTS9 Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis

Published OnlineFirst June 15, 2010; DOI: 10.1158/0008-5472.CAN-09-4510 Published OnlineFirst on June 15, 2010 as 10.1158/0008-5472.CAN-09-4510

Tumor and Stem Cell Biology Cancer Research Extracellular Protease ADAMTS9 Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis

Paulisally Hau Yi Lo1, Hong Lok Lung1, Arthur Kwok Leung Cheung1, Suneel S. Apte6, Kwok Wah Chan2, Fung Mei Kwong1, Josephine Mun Yee Ko1, Yue Cheng1, Simon Law3, Gopesh Srivastava2, Eugene R. Zabarovsky7, Sai Wah Tsao4, Johnny Cheuk On Tang2,5, Eric J. Stanbridge8, and Maria Li Lung1

Abstract ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC. Cancer Res; 70(13); OF1–10. ©2010 AACR.

Introduction fertility were shown (2). Moreover, altered expression of some ADAMTS genes has been shown in various cancers The a disintegrin-like and metalloproteinase (reprolysin and arthritis (1, 2). Three ADAMTS proteases (ADAMTS1, type) with thrombospondin type 1 motifs (ADAMTS)family ADAMTS8, and ADAMTS9) were previously shown to have consists of 19 secreted proteases having a well-defined do- antiangiogenic activity. ADAMTS1 and ADAMTS8 inhibited main structure. These enzymes consist of a prometalloprotei- vascular endothelial growth factor (VEGF)–induced angio- nase domain and a characteristic ancillary domain genesis as assayed by the chick chorioallantoic membrane containing one or more thrombospondin type 1 motifs (1). assay, suppressed fibroblast growth factor–induced vascular- Through analysis of mutant mice and human genetic disor- ization in the cornea pocket assay, and inhibited endothelial ders, the roles of ADAMTS in skin pigmentation, organogen- cell proliferation in vitro (3). ADAMTS9 was recently shown esis, limb development, connective tissue assembly, and to be a constitutive product of microvascular endothelial cells in both embryonic and adult mice and to act as a cell-autonomous angiogenesis inhibitor (4). Authors' Affiliations: Departments of 1Clinical Oncology and Center for Cancer Research, 2Pathology, 3Surgery, and 4Anatomy, University of The ability of a tumor to progress from a nonangiogenic to Hong Kong; 5Department of Applied Biology and Chemical Technology, an angiogenic phenotype is critical to cancer progression and Hong Kong Polytechnic University, Hong Kong SAR, People's Republic “ ” of China; 6Department of Biomedical Engineering, Lerner Research is termed the angiogenic switch (5). Expansion of a tumor Institute, Cleveland Clinic Foundation, Cleveland, Ohio; 7Department of mass beyond its initial microscopic size is dependent on the Microbiology, Tumor and Cell Biology, Department of Clinical Science recruitment of its own vascular supply, by angiogenesis and/ and Education, Södersjukhuset, Karolinska Institute, Stockholm, Sweden; and 8Department of Microbiology and Molecular Genetics, or blood vessel cooption (6–8). Failure of a tumor to recruit University of California, Irvine, California new microvascular endothelial cells or to reorganize the ex- Note: Supplementary data for this article are available at Cancer isting surrounding vasculature results in growth-limited, Research Online (http://cancerres.aacrjournals.org/). nonangiogenic tumors (9). Although related matrix metallo- P.H.Y. Lo and H.L. Lung contributed equally to this work. proteases (e.g., ADAM and ADAMTS proteases) have been Corresponding Author: Maria Li Lung, Department of Clinical Oncology, implicated in tumor progression and angiogenesis, the spe- The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, People's Republic of China. Phone: 852-2819-9783; Fax: 852- cific role of ADAMTS9 in tumor angiogenesis is less clearly 2819-5872; E-mail: [email protected]. defined. Our previous functional genomic studies show that doi: 10.1158/0008-5472.CAN-09-4510 ADAMTS9 is associated with tumor suppression in two aero- ©2010 American Association for Cancer Research. digestive tract cancers, namely esophageal squamous cell

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carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Time PCR System (Applied Biosystems). The primers used Downregulation of ADAMTS9 expression was commonly ob- for semiquantitative PCR are listed in Supplementary Table served in tumor tissues and cell lines of both cancers. Pro- S1. All PCR assays were performed in triplicate in two inde- moter hypermethylation contributes to ADAMTS9 gene pendent experiments. For the analysis of mRNA stability of MMP9 and VEGFA, the transcription inhibitor, actinomycin silencing in both ESCC and NPC (10, 11). Importantly, previ- D (Sigma-Aldrich, 5 μg/mL; ref. 17), was added to ADAMTS9 ous studies indicate that ADAMTS9 protein expression in stable transfectants. NPC is significantly associated with lymph node metastases (11). The role of this protein in cancer development remains Western blot analysis unclear. In the present study, we investigated the in vivo and Western blot analysis of ADAMTS9 was performed as pre- in vitro functional roles of ADAMTS9 in angiogenesis and viously reported (18). The ADAMTS9 propeptide domain tar- ESCC and NPC tumorigenesis. Antiangiogenic and tumor- geting antibody (Abcam) and Ab-1 (Calbiochem) were used suppressive activities of ADAMTS9 were studied by stringent as primary antibodies for the detection of ADAMTS9 and α- in vivo tumorigenicity and Matrigel plug angiogenesis assays. tubulin, respectively. The effects of conditioned medium from ADAMTS9 stable ADAMTS9 transfectants were assessed in in vitro tube formation ability Stable transfection of To generate stable clones, which express wild-type assays using human umbilical vein endothelial cells (HUVEC) ADAMTS9 in ESCC and NPC cell lines, KYSE30 and HONE1- to better understand its role in this important process. 2cellsweretransfectedwithpCR3.1-ADAMTS9 and pETE- Bsd-ADAMTS9, respectively, as previously reported (11, 18). Materials and Methods Knockdown of ADAMTS9 in MCH8.12 cells Cell lines and culture conditions ADAMTS9 knockdown was achieved by using BLOCK-iT The ESCC cell line KYSE30, obtained from Deutsche Pol II miR RNAi Expression Vector Kit (Invitrogen), and Sammlung von Mikroorganismen und Zellkulturen GmbH the sequences of the pair of the shRNA oligonucleotides (12), and immortalized esophageal epithelial cell line NE1 are 5′-TGCTGTCACCAGCCAGGTTAATCCTTGTTTTGGC- were cultured as previously described (10). Stable ESCC CACTGACTGACAAGGATTACTGGCTGGTGA-3′ and ADAMTS9 transfectants (EC-AD clones) and pCR3.1 vector- 5′-CCTGTCACCAGCCAGTAATCCTTGTCAGTCAGTGGC- alone control (EC-V clone) were cultured in medium contain- CAAAACAAGGATTAACCTGGCTGGTGAC-3′, which target at ing 400 μg/mL neomycin. The recipient NPC HONE1 cell line nucleotide positions 770 to 780 of the human ADAMTS9 cDNA and the previously established HONE1/chromosome 3 micro- (NM_182920). In brief, pcDNA6.2GM-shRNA770 plasmid with cell hybrid (MCH) cell line MCH8.12 were used for the the ADAMTS9 shRNA oligonucleotide or the vector-alone ADAMTS9 knockdown analysis. MCH8.12 contains an extra- pcDNA6.2-GW/EmGFP-miR (pcDNA6.2GM) plasmid was truncated chromosome 3 (deleted at 3p24) transferred by stably transfected into the recipient cell line, MCH8.12, which microcell-mediated chromosome transfer to the recipient strongly expresses ADAMTS9 (11). HONE1 cell; it exhibits a prolonged latency period before tumor formation. HONE1 and MCH8.12 were maintained as pre- Tumorigenicity assay and tumor segregant analysis viously described (13). Stable ADAMTS9 knockdown clones The cell lines were injected subcutaneously into three 6- to were maintained in culture medium containing 500 μg/mL 8-week-old female athymic BALB/c Nu/Nu mice. Subcutane- neomycin and 5 μg/mL blasticidin. The immortalized naso- ous injection and preparation of tumor segregants were per- pharyngeal epithelial cell line NP460 was cultured as described formed as previously described (10, 19). In brief, 5 × 106 and (14). Construction of a pETE-Bsd–responsive vector and a 1×107 cells were injected into both flanks of three nude mice HONE1 cell line, HONE1-2, producing the tetracycline transac- (six sites) for each ESCC and NPC cell line, respectively. The tivator tTA, was described by Protopopov and colleagues (15). tumor sizes were measured weekly. Tumors arising from Stable NPC transfectants with ADAMTS9 transgene (NPC-AD nonsuppressing ADAMTS9 transfectants were subsequently clones) or with pETE-Bsd vector-alone (NPC-V clone) were excised and reconstituted into tissue culture. These are the maintained in culture medium containing 500 μg/mL neomy- tumor segregant cell lines used for further analysis. For inhi- cin and 5 μg/mL blasticidin. HUVECs (Lonza) were cultured bition of the tetracycline-inducible expression of ADAMTS9 in as previously described (16). All cultures were regularly mon- NPC transfectant cell lines in vivo, 200 μg/mL doxycycline itored for Mycoplasma contamination and were uniformly was added to the drinking water of mice 1 week before injec- negative. tion; water containing doxycycline was changed twice a week.

Reverse transcription-PCR and real-time quantitative HUVEC tube formation assay reverse transcription-PCR analyses Conditioned media were collected by incubating Semiquantitative and quantitative PCR were performed as ADAMTS9 and vector-alone ESCC and NPC transfected cells previously reported (10, 11). Real-time quantitative PCRs with Dulbecco's modified Eagle's medium (DMEM) without were performed using ADAMTS9 and GAPDH Taqman probes serum for 24 hours. For the NPC ADAMTS9 and vector-alone or the SYBR Green PCR master mix in a StepOnePlus Real- transfectants, conditioned media ± 0.2 μg/mL doxycycline

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were obtained. A total of 4 × 104 HUVEC cells were seeded performed according to the manufacturer's instructions. Ab- into each well coated with 50 μL Matrigel (BD Biosciences) sorbance was detected by Labsystems Multiskan MS Plate and incubated with 100 μL conditioned media from vector- Reader (Thermo Fisher Scientific, Inc.). The assay was per- alone and ADAMTS9 transfectants plus 1% fetal bovine se- formed in three independent experiments. rum. Cells were then incubated for 5 hours to allow formation of tube-like structures (16). The images at 100× magnification Statistical analysis were captured using an inverted microscope (Nikon Instru- Statistical analysis was performed using SPSS11.0 statistics ments, Inc.). Total tube length was measured and compared calculation software (SPSS, Inc.). Comparisons between for three different viewing fields by the SPOT software (Diag- ADAMTS9 and vector-alone transfectants in all experiments nostic Instruments). The primary ADAMTS9 targeting anti- were performed by Student's t test. A P value of <0.05 was body (Abcam) was used as a neutralizing antibody for considered as significant. blocking the effects of the extracellular ADAMTS9 protein in the conditioned media. Another irrelevant rabbit polyclon- Results al antibody was used as a negative control immunoglobulin. Activation of ADAMTS9 expression suppresses In vivo Matrigel plug angiogenesis assay tumor formation in vivo A total of 5 × 106 ESCC cells or 1 × 107 NPC cells in 50 μL ADAMTS9 expression is downregulated in the cell lines DMEM mixed with 250 μL ice-cold Matrigel (BD Biosciences) KYSE30 and HONE1 derived from ESCC and NPC, respective- were subcutaneously injected into nude mice. Each cell line ly (10, 11); therefore, they were chosen for the stable was injected into one site for five nude mice. Matrigel con- ADAMTS9 transfection and the subsequent functional analy- taining the cell suspension polymerized after injection and ses. As shown in Fig. 1A and B, gene and protein expression formed a plug impregnated with tumor cells. The gel plugs of ADAMTS9 was induced in both ESCC ADAMTS9 transfec- were removed after 7 days, fixed with formalin, and embed- tants, EC-AD7 and EC-AD9, which were analyzed by quanti- ded in paraffin. Histologic sections were stained with hema- tative real-time PCR and Western blot analyses, respectively. toxylin and eosin and endothelial cell marker anti-CD34 ADAMTS9 expression in four NPC transfectants, NPC-AD1, monoclonal antibody (Santa Cruz Biotechnology). The slides NPC-AD20, NPC-AD23, and NPC-AD25, was induced in the were incubated with the anti-CD34 antibody (1:40 dilution) absence of doxycycline (Fig. 1A and B). Doxycycline treat- for immunohistochemistry as previously described (18). ment inactivates the tTA transcriptional activator, resulting CD34-positive staining of vascular endothelial cells was ana- in reduction of transgene and protein expression. Reduction lyzed by ImageScope v10 software (Aperio). of ADAMTS9 transcript and protein levels was observed following doxycycline treatment in the HONE1-2 stable clones Human angiogenesis antibody array (Fig. 1A and B). High gene and protein expression levels were Conditioned media were obtained as previously described. detected in NPC-AD1, NPC-AD20, NPC-AD23, and NPC-AD25 Proteins in conditioned media were hybridized with a hu- cell lines [without doxycycline treatment (−dox)]; ADAMTS9 man angiogenesis antibody array dotted with 43 human levels only dropped to those observed in the vector-alone angiogenesis–related antibodies (RayBiotech). The assay control with NPC-AD25 [with doxycycline treatment (+dox); was performed as described in the manufacturer's manual. Fig. 1A and B]. Recipient KYSE30 and HONE1 cells are highly tumorigenic Gelatin zymography and formed palpable tumors of 150 mm3 1 week after injec- Matrix metalloproteinase 9 (MMP9) protein expression tion in all six injection sites. When ADAMTS9 was expressed was measured by gelatin zymography and was performed in EC-AD9 and NPC-AD25 (−dox), prolonged latency periods as previously described (20). In brief, conditioned medium for tumor formation of up to 4 to 6 weeks were observed, was mixed with loading buffer without β-mercaptoethanol, which was significantly longer than the 1- to 2-week latency and loaded onto a 10% SDS-PAGE gel with 0.1% gelatin. After period observed with vector-alone clones (Fig. 1C; Table 1). the samples were fractionated, the gel was washed twice with When ADAMTS9 expression was reduced in NPC-AD25 2.5% Triton X-100 and then incubated at 37°C with reaction (+dox), the average tumor size was 883 mm3, whereas when buffer [50 mmol/L Tris-Cl (pH 7.5), 5 mmol/L CaCl2,and transcription was switched on, the average size was reduced 3 0.02% NaN3] overnight. The gel was stained with 0.1% Coomas- to 149 mm 4 weeks after injection. The difference was sta- sie brilliant blue R-250 (Sigma-Aldrich). MMP9 activity was tistically significant (P = 0.02). These results suggest that visualized as a clear band on a blue background with a size ADAMTS9 expression by tumor potently suppresses tumor of 92 kDa. The assay was performed in three independent ex- formation in both ESCC and NPC in vivo. periments. Quantitation of the MMP band was performed by using the Quantity One Gel Documentation System (Bio-Rad). Loss of expression of ADAMTS9 in tumors and a tumor segregant derived from tumorigenic transfectants is Human VEGF immunoassay associated with tumorigenicity VEGFA in conditioned media secreted by various ESCC In vivo tumorigenicity results showed that there is no signif- and NPC cell lines was detected by the Quantikine Human icant tumor suppression for the other ADAMTS9 stable trans- VEGF Immunoassay system (R&D Systems). The assay was fectants, EC-AD7 and NPC-AD1, NPC-AD20, and NPC-AD23

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Figure 1. A, real-time quantitative RT-PCR analysis of ADAMTS9 in ESCC and NPC ADAMTS9 stable clones. Relative ddCt of each clone was compared with its respective vector-alone control. dox, doxycycline. B, Western blot analysis of the ADAMTS9 protein in stable clones. α-Tubulin was used as an internal control. C, tumor growth kinetics of ESCC and NPC ADAMTS9 and vector-alone transfectants. Each data point represents an average tumor volume at six sites inoculated for each cell line.

(−dox), when compared with the vector-alone transfectants, tional inactivation of ADAMTS9 during tumorigenesis in nude EC-V1 and NPC-V1 (−dox; Fig. 1C; Table 1). To check the status mice contributes to the lack of tumor suppression of EC-AD7, of ADAMTS9 following tumor formation in these tumorigenic NPC-AD1, NPC-AD20, and NPC-AD23. ADAMTS9 clones, total RNA was isolated from excised tumors (EC-AD7-T1-T3, NPC-AD1-T1-T6, NPC-AD20-T1-T3, NPC- Tumorigenicity restored by ADAMTS9 knockdown in a AD23-T1-T6), and cells from one tumor (NPC-AD1-T6-TS) microcell hybrid cell line were reconstituted in culture medium. Eight primer pairs To further confirm the tumor-suppressive effect of spanning the ADAMTS9 transcript were used to analyze the ADAMTS9, ADAMTS9 knockdown in the nontumorigenic regional loss of ADAMTS9 gene expression in those tumors HONE1/chromosome 3 MCH8.12, which expresses high levels and the tumor segregants by RT-PCR of the 7,335-bp of ADAMTS9 mRNA (11), was performed. Stable ADAMTS9 ADAMTS9 transcript. In general, the NPC-AD1 series of tumors knockdown transfectants were obtained by transfecting and its tumor segregants showed an extensive deletion in the pcDNA6.2GM-shRNA770 into MCH8.12. Reduction of ADAMTS9 mRNA. The other tumors derived from the EC-AD7 ADAMTS9 protein expression was observed in the stable and NPC-AD20 and NPC-AD23 showed smaller discrete dele- clones, 8.12-A9-shRNA770-N59 and 8.12-A9-shRNA770-N60 tions. RT-PCR analysis using the primers p4 and p5 consistent- (Fig. 2C), when compared with the vector-alone 8.12- ly showed the loss of ADAMTS9 transcript in all tumors and pcDNA-V1. The specificity of the shRNA oligonucleotide the tumor segregant; a common minimum region of deletion was tested; RT-PCR results show that gene expression of was observed in the third and fourth thrombospondin type 1 ADAMTS1 and ADAMTS8 was not affected in stable repeats of ADAMTS9 (Fig. 2A). Representative RT-PCR results ADAMTS9 knockdown transfectants 8.12-A9-shRNA770-N59 with and without loss of ADAMTS9 transcript regions in the and 8.12-A9-shRNA770-N60 (data not shown). Tumorigenici- selected tumors are shown in Fig. 2B. We conclude that func- ty was suppressed in the vector-alone clone 8.12-pcDNA-V1,

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and tumors were observed at only two sites of injection for vector-alone controls (Fig. 3B). The tube-forming ability of up to 8 weeks after inoculation (Fig. 2D; Table 1). Tumors HUVEC cells was significantly decreased to 66.4%, 61.1%, appeared with both ADAMTS9 knockdown clones in at least and 61.5% after incubation with EC-AD7 and EC-AD9 and four injection sites, which showed similar growth kinetics. NPC-AD25 (−dox) cell conditioned media, respectively, as The differences were statistically significant when compared compared with the vector-alone controls (Fig. 3C). The inhib- with the vector-alone controls (P = 0.01 and 0.003, respective- itory effect on tube formation was restored to control levels ly). When compared with the tumor growth rate of HONE1 in NPC-AD25 (+dox) when transgene expression was re- cells, the two ADAMTS9 knockdown MCHs were significantly pressed (Fig. 3B and C). To further validate the specific inhibi- slower (P = 0.02 and 0.016, respectively; Fig. 2D; Table 1). tory effect of secreted ADAMTS9 protein in the conditioned media, ADAMTS9-neutralizing antibody was added together ADAMTS9 reduces tube formation by HUVEC in vitro with the conditioned medium to block its activities; reduction The “angiogenic switch” is critical for solid tumor forma- in the activities of secreted ADAMTS9 subsequently decreased tion (5), and ADAMTS9 was shown to suppress tumor forma- the tube-forming ability of the three ESCC/NPC ADAMTS9 tion in ESCC and NPC in the current study. This study transfectants (Fig. 3B and C). The ADAMTS9-neutralizing examines the antiangiogenic property of ADAMTS9 and antibody had no significant effect on tube formation in the how it can contribute to its tumor-suppressive function. HU- vector-alone clones (Fig. 3B and C). An irrelevant negative VEC tube formation assay was used to test the effect of control antibody has no significant effect on the tube forma- ADAMTS9 on angiogenesis in vitro. ADAMTS9 is a secreted tion in the ADAMTS9 transfectants (data not shown). protein associated with the cell surface, but has also been shown to be present in the conditioned medium of expres- ADAMTS9 inhibits in vivo angiogenesis sing cells (4). Concentrated conditioned media collected The antiangiogenic activity of ADAMTS9 in vivo was as- from both ESCC and NPC ADAMTS9 transfectants were an- sessed in mice with the Matrigel plug assay. The tumor- alyzed by Western blotting to show the presence of secreted suppressive ESCC and NPC transfectants, EC-AD9 and ADAMTS9 proteins compared with vector-alone transfec- NPC-AD25, were used, as the ADAMTS9 transgenes of these tants, EC-V1 and NPC-V1 (Fig. 3A). Secreted ADAMTS9 pro- clones are stable in in vivo conditions. The anti-CD34 anti- tein expression was significantly reduced in NPC-AD25 body microvessel–stained gel plugs were analyzed; represen- (+dox; Fig. 3A). Conditioned media from EC-AD7 and EC- tative results are shown in Fig. 4A and B (top panels). The AD9 and NPC-AD25 (−dox) inhibited formation of tube-like numbers of microvessels of both ESCC and NPC clones, structures of HUVEC cells, as compared with their respective EC-AD9 and NPC-AD25 (−dox), were substantially reduced

Table 1. Tumorigenicity assays of ADAMTS9 transfectants and ADAMTS9 knockdown MCHs

Cell line Identification Dox Tumor formation Latency period (wk) P*†‡§ (no. tumors/no. for tumor volume sites) of 150 mm3

EC-V1 KYSE30× pCR3.1 − 6/6 1–2 − EC-AD7 KYSE30× pCR3.1-ADAMTS9 − 6/6 1 0.71* EC-AD9 KYSE30× pCR3.1-ADAMTS9 − 3/6 4–6 0.002* NPC-V1 HONE1-2× pETE-Bsd − 6/6 1–2 − + 6/6 1–2 0.4† NPC-AD1 HONE1-2× pETE-Bsd-ADAMTS9 − 6/6 1–2 0.14* NPC-AD20 HONE1-2× pETE-Bsd-ADAMTS9 − 4/6 1–4 0.35* NPC-AD23 HONE1-2× pETE-Bsd-ADAMTS9 − 5/6 1–4 0.23* NPC-AD25 HONE1-2× pETE-Bsd-ADAMTS9 − 5/6 4–6 0.01* + 5/6 1–4 0.45*, 0.02† HONE1 Parental NPC cell line − 6/6 1–3 − 8.12-pcDNA-V1 8.12 × pcDNA6.2GM − 2/6 NA 0.004‡ 8.12-A9-shRNA770-N59 8.12 × ADAMTS9-shRNA770 − 4/6 4–7 0.01‡, 0.02§ 8.12-A9-shRNA770-N60 8.12 × ADAMTS9-shRNA770 − 5/6 4–6 0.003‡, 0.016§

Abbreviations: Dox, doxycycline; NA, not applicable. *P value obtained by comparison with EC-V1/NPC-V1 (−dox). †P value obtained by comparison between tumor sizes ± dox. ‡P value obtained by comparison with HONE1. §P value obtained by comparison with pcDNA6.2GM.

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Figure 2. A, RT-PCR analysis of ESCC and NPC tumorigenic ADAMTS9 stable clone-derived tumors and tumor segregants with eight primer pairs covering the entire ADAMTS9 transcript. Relative localizations of the primer pairs are indicated. ○, positive expression; •, no expression; , reduced expression. B, representative RT-PCR results of the ADAMTS9 transcript regions P4 and P6 in selected tumors derived from stable ADAMTS9 clones. GADPH served as a loading control. C, Western blot analysis of ADAMTS9 knockdown transfectants in two NPC clones and the vector-alone control. D, tumor growth kinetics of the two ADAMTS9 knockdown and vector-alone clones and HONE1 cells.

to 58.8% and 46.9%, respectively, of the vector-alone controls macrophage colony-stimulating factor (GM-CSF), and (Fig. 4A and B, bottom panels). Repression of the transgene transforming growth factor-β1 (TGF-β1) was observed in in NPC-AD25 (+dox) restored the angiogenesis levels ob- ADAMTS9 transfectants (data not shown). Real-time quan- served in the gel plug to that of the vector-alone control titative PCR was used to confirm their expression in both (Fig. 4B). In addition, cell necrosis was frequently observed ESCC and NPC transfectants, EC-AD7 and EC-AD9 and in the central regions of tumor nodules in the gel plug of NPC-AD1, NPC-AD20, NPC-AD23, and NPC-AD25. MMP9 NPC-AD25 (−dox; Fig. 4C). This is possibly connected with gene expression was consistently decreased in all ADAMTS9- poorer vasculature of the tumor cells in these plugs. expressing ESCC and NPC clones (Fig. 5A). After transgene repression in NPC clones, MMP9 gene expression was higher Downregulation of MMP9 and VEGFA gene and protein in all four NPC transfectants (+dox), as compared with the expression in media derived from ADAMTS9 clones expressing ADAMTS9 (−dox). We further used gelatin stable transfectants zymography to detect and quantify the MMP9 protein in con- We used an antibody array containing several angiogenesis- ditioned media. Strong MMP9 signals were observed in the related antibody probes to study the proteins associated vector-alone EC-V1 and NPC-V1 (Fig. 5B). When ADAMTS9 with the antiangiogenic activities of ADAMTS9.Condi- was overexpressed in EC-AD9 and NPC-AD25 (−dox), MMP9 tioned media from the ESCC ADAMTS9 and vector-alone signals were significantly reduced. MMP9 activity was transfectants were collected and hybridized to this anti- restored when ADAMTS9 expression was suppressed in body array to determine differential expression levels of NPC-AD25 (+dox). the angiogenesis-related proteins. Downregulated expres- On the other hand, real-time PCR results show that sion of VEGFA, MMP9, interleukin 8 (IL-8), granulocyte VEGFA-189 gene expression decreased in the two ESCC

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clones (EC-AD7 and EC-AD9) and three of four NPC clones Discussion (NPC-AD1, NPC-AD23, and NPC-AD25; Fig. 5A). The other angiogenesis-related genes, IL-8, TGF-β1, and GM-CSF, were Functional analyses of ADAMTS9 strongly support its less consistently downregulated in the ESCC and NPC clones important role in vivo and in vitro. Using inducible and (data not shown). To quantitate VEGFA protein expression, constitutive gene expression systems, in vivo and in vitro an- we used enzyme-linked immunosorbent assay (ELISA) to de- giogenesis and tumorigenicity assays clearly show that the tect VEGFA secreted by ADAMTS9-overexpressing transfec- overexpression of ADAMTS9 is sufficient to induce potent tants in the conditioned media. Figure 5C shows that suppression of tumor formation and angiogenesis in both VEGFA protein expression was reduced to 55% and 17% in ESCC and NPC. Importantly, by knockdown of ADAMTS9 ex- EC-AD9 and NPC-AD25 (−dox), as compared with their pression in a nontumorigenic MCH, we show that ADAMTS9 vector-alone controls, respectively. VEGFA protein expres- knockdown clones revert to the tumorigenic phenotype of sion was restored in NPC-AD25 (+dox) when the ADAMTS9 parental cells. Gene and protein expression analyses of gene expression was switched off. ADAMTS9 transfectants revealed that its reduced expression To investigate the regulatory mechanism of MMP9 and is associated with the transcriptional regulation of proangio- VEGF by ADAMTS9, NPC-AD25 was treated with actinomycin genic factors, MMP9 and VEGFA,inbothESCCandNPC. D for 24 hours to determine whether transcription or mRNA Hence, the data strongly suggest that ADAMTS9 plays a crit- stability of MMP9 and VEGFA would be affected. Figure 5D ical role in the angiogenic switch and transforms both ESCC shows that the MMP9 and VEGFA-189 transcripts were still and NPC cell lines from a proangiogenic to a nonangiogenic stable after treatment with actinomycin D. The increased ex- phenotype. Inhibition of tumor angiogenesis is a common pression of MMP9 and VEGFA-189 after the addition of doxy- mechanism for tumor suppression in both tumor types. cycline was reduced to less than basal levels when the cell Based on the deletion patterns of the ADAMTS9 transcript line was treated with actinomycin D. Hence, it is likely that in tumors and a tumor segregant derived from the tumori- transcription of both MMP9 and VEGFA-189, and not their genic transfectants, it is tempting to speculate that the stability, was regulated by ADAMTS9. tumor-suppressive activity of ADAMTS9 in ESCC and NPC

Figure 3. A, Western blot analysis of conditioned medium from the ADAMTS9 and vector-alone stable clones. Total protein staining by Coomassie blue was used to indicate equal loading. B, representative results of HUVEC tube formation assay of the ADAMTS9 and vector-alone transfectants and after treatment with the ADAMTS9-neutralizing antibody. C, percentage of tube formation ability of ADAMTS9 clones with or without the ADAMTS9-neutralizing antibody, as compared with their corresponding vector-alone controls. *, P < 0.05, statistically significant difference from the vector-alone clone.

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Figure 4. Representative results from the Matrigel plug assay in ESCC (A) and NPC (B). Endothelial cells were stained with anti-CD34 antibody, as indicated by arrows. CD34 staining index of ADAMTS9 transfectants as compared with their corresponding vector-alone controls. *, P < 0.05, statistically significant difference from the vector-alone clone. C, representative image of cell necrosis (acellular areas indicated by *) observed in the center region of tumor nodules formed by the NPC ADAMTS9 transfectant NPC-AD25 clone (−dox). H&E, hematoxylin and eosin.

is associated with the thrombospondin (TSP) domains in the tumor segregant derived from tumorigenic clones. This kind of COOH-terminal region of the gene. functional inactivation due to elimination of transgene overex- Our findings strongly suggest that the antiangiogenic ac- pression was observed in our previous studies of the tumor- tivities of ADAMTS9 play the critical role of tumor suppres- suppressor gene cell adhesion molecule 1 (CADM1, formerly sion in both ESCC and NPC. These findings are consistent called TSLC1) and THY1 in NPC (19, 21). The in vitro angiogen- with recent findings in which the ADAMTS9 protein ex- esis assay results show that EC-AD7 could significantly suppressed by the microvascular endothelial cells was shown press tube formation of HUVEC cells, as the ADAMTS9 to be antiangiogenic in both the Adamts9+/− mice and the transgene is stable and remains activated in in vitro conditions. siRNA knockdown of cultured human microvascular endo- The present functional studies were also performed using thelial cells. In contrast, unlike ADAMTS1, which exhibits the stable NPC MCH8.12 ADAMTS9 knockdown clones. Both its antiangiogenic effects by cleavage of TSPs and sequestra- clones showing reduced ADAMTS9 expression clearly tion of VEGFA-165, ADAMTS9 neither cleaves TSP-1 and reverted back to their tumorigenic phenotype. However, be- TSP-2, nor binds VEGFA-165 (4). A key conclusion of those cause tumor growth kinetics was still lower than that of the studies was the cell-autonomous effect of ADAMTS9 in endo- original recipient HONE1 cells, it is possible that other thelial cells. The present studies highlight a non–cell auton- growth-inhibitory gene(s) besides ADAMTS9 might still be omous mechanism by which ADAMTS9 produced by tumor present on chromosome 3. Previous studies indicate that cells has an effect on angiogenesis; thus, ADAMTS9 may act PTPRG and RASSFIA and BLU, at the nearby 3p14-21 and on endothelial cells through a dual mechanism. 3p21.3 regions, respectively, are identified as tumor-suppressor For the tumorigenic ADAMTS9 transfectants, EC-AD7, NPC- genes in NPC (22–25). AD1, NPC-AD20, and AD-23 (−dox), tumors formed 1 week af- Except for the initial association of microvascular endo- ter injection. After inoculation of these clones in nude mice, thelial cells expressing ADAMTS9 with angiogenesis (4), both reduction and loss of the region encoding the third and the antiangiogenic activities of ADAMTS9 in human can- the fourth thrombospondin type 1 repeats were observed by cer cells have yet to be reported. The results of these RT-PCR analysis of mRNA in all tumors and the representative studies thus provide clear evidence for the importance

OF8 Cancer Res; 70(13) July 1, 2010 Cancer Research

Tumor-Suppressive and Antiangiogenic ADAMTS9

of ADAMTS9 in angiogenesis and tumor development in levels of the proangiogenic factors MMP9 and VEGFA. two important malignancies, ESCC and NPC. Our results In future studies, it will be important to define the precise suggest that ADAMTS9 inhibition of angiogenesis in both relationship between ADAMTS9 and these two key regula- cancers is associated with reduction of gene expression tors of angiogenesis.

Figure 5. A, real-time quantitative PCR analysis of MMP9 and VEGFA-189 in ESCC and NPC ADAMTS9 transfectants. Fold change of the relative gene expression of MMP9 and VEGFA-189 of each clone was compared with their corresponding vector-alone transfectants. Relative ddCt of the ADAMTS9 gene expression of each clone was compared with that of the corresponding vector-alone transfectants. B, zymography of conditioned media from ESCC and NPC ADAMTS9 stable transfectants. Average MMP9 activities were calculated from three independent experiments. Fold change of MMP9 activities of each clone was compared with that of the vector-alone controls. Representative zymography results are shown on the right. MMP9 and MMP2 are indicated. C, quantitative analysis of human VEGFA in conditioned media by ELISA. Relative VEGFA expression in conditioned medium from ADAMTS9 stable transfectants was compared with their vector-alone controls. *, P < 0.05, statistically significant difference from the vector-alone clone. D, real-time quantitative PCR analysis of MMP9 and VEGFA-189 in NPC-AD25 (±dox) after actinomycin D treatment. Fold change of the relative gene expression of MMP9 and VEGFA-189 in each of the treated cells was compared with that of the untreated cells.

www.aacrjournals.org Cancer Res; 70(13) July 1, 2010 OF9

Lo et al.

Disclosure of Potential Conflicts of Interest Research Council, Swedish Institute, Cancer Research Institute in New York/Concern Foundation in Los Angeles, and Karolinska Institute (E.R. Zabarovsky). No potential conflicts of interest were disclosed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China: grants HKU6617/08M and HKU6415/06M (M.L. Received 12/11/2009; revised 04/14/2010; accepted 04/28/2010; published Lung); NIH award AR49930 (S.S. Apte); and Swedish Cancer Society, Swedish OnlineFirst 06/15/2010.

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OF10 Cancer Res; 70(13) July 1, 2010 Cancer Research

Extracellular Protease ADAMTS9 Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis

Paulisally Hau Yi Lo, Hong Lok Lung, Arthur Kwok Leung Cheung, et al.

Cancer Res Published OnlineFirst June 15, 2010.

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