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Cyr61 Mediates Hepatocyte Growth Factor–Dependent Tumor Cell Growth, Migration, and Akt Activation

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Cyr61 Mediates Hepatocyte Growth Factor–Dependent Tumor Cell Growth, Migration, and Akt Activation Published OnlineFirst March 16, 2010; DOI: 10.1158/0008-5472.CAN-09-3570 Tumor and Stem Cell Biology Cancer Research Cyr61 Mediates Hepatocyte Growth Factor–Dependent Tumor Cell Growth, Migration, and Akt Activation C. Rory Goodwin1,3, Bachchu Lal1,2, Xin Zhou1, Sandra Ho1, Shuli Xia1, Alexandra Taeger1, Jamie Murray1, and John Laterra1,2,3,4 Abstract Certain tumor cell responses to the growth factor–inducible early response gene product CCN1/Cyr61 overlap with those induced by the hepatocyte growth factor (HGF)/c-Met signaling pathway. In this study, we investigate if Cyr61 is a downstream effector of HGF/c-Met pathway activation in human glioma cells. A semiquantitative immunohistochemical analysis of 112 human glioma and normal brain specimens showed that levels of tumor-associated Cyr61 protein correlate with tumor grade (P < 0.001) and with c-Met protein expression (r2 = 0.4791, P < 0.0001). Purified HGF rapidly upregulated Cyr61 − mRNA (peak at 30 minutes) and protein expression (peak at 2 hours) in HGF /c-Met+ human glioma cell lines via a transcription- and translation-dependent mechanism. Conversely, HGF/c-Met pathway in- hibitors reduced Cyr61 expression in HGF+/c-Met+ human glioma cell lines in vitro and in HGF+/c-Met+ glioma xenografts. Targeting Cyr61 expression with small interfering RNA (siRNA) inhibited HGF-induced cell migration (P < 0.01) and cell growth (P < 0.001) in vitro. The effect of Cyr61 on HGF-induced Akt pathway activation was also examined. Cyr61 siRNA had no effect on the early phase of HGF-induced Akt phosphorylation (Ser473) 30 minutes after stimulation with HGF. Cyr61 siRNA inhibited a second phase of Akt phosphorylation measured 12 hours after cell stimulation with HGF and also inhibited HGF-induced phosphorylation of the Akt target glycogen synthase kinase 3α. We treated preestablished subcutaneous glioma xenografts with Cyr61 siRNA or control siRNA by direct intratumoral delivery. Cyr61 siRNA inhibited Cyr61 expression and glioma xenograft growth by up to 40% in a dose-dependent manner (P < 0.05). These results identify a Cyr61-dependent pathway by which c-Met activation mediates cell growth, cell migration, and long-lasting signaling events in glioma cell lines and possibly astroglial malignancies. Cancer Res; 70(7); 2932–41. ©2010 AACR. Introduction mor xenograft formation, growth rates, and angiogenesis in many cancer model systems (6–9). The link between Aberrations in receptor tyrosine kinase (RTK) systems c-Met and malignant progression has stimulated the activate a multitude of oncogenic mechanisms that in- search for downstream mediators of these responses, in crease overall malignancy. The RTK c-Met and its multi- hope of understanding c-Met signaling and identifying functional ligand hepatocyte growth factor (HGF) are new potential therapeutic targets. overexpressed or hyperactivated in many solid neoplasms c-Met is a potent activator of multiple signaling path- and correlate with poor patient survival in select malig- ways, such as phosphatidylinositol 3-kinase (PI3K)/Akt, nancies. HGF/c-Met pathway activation can induce a Ras/mitogen-activated protein kinase, signal transducer variety of cell type–dependent and context-dependent bio- and activator of transcription (STAT), and NFκB, which ac- logical responses, such as migration/invasion, proliferation, tivate a wide variety of oncogenic targets (10, 11). Expres- angiogenesis, and cytoprotection (1–5). Inhibiting HGF sion microarray analysis of human glioblastoma cells and/or c-Met expression or function potently inhibits tu- stimulated with HGF resulted in the identification of sev- eral genes that are upregulated by c-Met activation. One HGF-responsive gene identified is the cysteine-rich 61/con- Authors' Affiliations: 1Kennedy Krieger Institute and Departments of nective tissue growth factor/nephroblastoma overexpressed 2Neurology, 3Neuroscience, and 4Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland (CCN) family member Cyr61 (12). Cyr61 is a secreted Note: Supplementary data for this article are available at Cancer heparin-binding protein that associates with the cell sur- Research Online (http://cancerres.aacrjournals.org/). face and extracellular matrix (13–15). Cyr61, which is nor- Corresponding Author: John Laterra, Kennedy Krieger Research Insti- mally involved in wound healing, chondrogenesis, and tute, 707 North Broadway, Baltimore, MD 21205. Phone: 443-923- neovascularization in vivo, is a growth factor–inducible and 2679; Fax: 443-923-2695; E-mail: [email protected]. hypoxia-inducible early response gene that interacts with doi: 10.1158/0008-5472.CAN-09-3570 various cellular integrins to induce the expression of a diverse ©2010 American Association for Cancer Research. array of genes (14–18). Purified Cyr61 protein has been 2932 Cancer Res; 70(7) April 1, 2010 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 16, 2010; DOI: 10.1158/0008-5472.CAN-09-3570 Cyr61 and c-Met Signaling Responses shown to regulate cell adhesion, stimulate chemotaxis, i.p. injection of ketamine (100 mg/kg) and xylazine (5 mg/kg). augment growth factor–induced DNA synthesis, foster cell For subcutaneous xenografts, nu/nu mice received 4 × 106 survival, and enhance angiogenesis in vivo (19, 20). Cyr61 cells in 0.05 mL of plain medium s.c. in the dorsal flank. expression has been linked to poor outcomes in a multitude When tumors reached 200 mm3, the mice were randomly di- of solid tumors (21, 22), is implicated in increased tumori- vided into groups (n = 5 per group) and received the indicat- genicity, and is overexpressed in invasive breast cancer and ed doses of either L2G7 or isotype-matched control mAb astrocytoma cell lines (23–28). Moreover, forced expression (5G8) in 0.1 mL PBS i.p. as previously described (30). Tumor of Cyr61 in a low-grade U343 glioma model markedly volumes were estimated by measuring two dimensions enhanced tumorigenicity and vascularization in vivo.Xie [length (a)andwidth(b)] and calculating volume as V = and colleagues (20) showed that multiple integrins, espe- ab2/2 (30, 31). At the end of each experiment, tumors were α β cially v 3, were upregulated by Cyr61 protein along with excised and frozen in liquid nitrogen, and protein was ex- an increase in integrin-linked kinase (ILK) activity and tracted for immunoblot analysis. Akt activation. For intracranial xenografts, Scid/beige mice received 1 × The overlapping phenotypic effects of c-Met activation and 105 cells/2 μL by stereotaxic injection into the right cau- Cyr61 suggest a novel mechanism by which the HGF/c-Met date/putamen (29). L2G7 or 5G8 mAb was administered pathway influences cell behavior. This study investigates as above. Groups of mice (n = 5) were sacrificed by perfu- whether the HGF/c-Met pathway and Cyr61 are correlated sion fixation at the indicated times, and the brains were re- in human gliomas and whether HGF induces Cyr61 in glioma moved for histologic studies. Vibratome/microtome models. Furthermore, we investigate if inhibiting Cyr61 in perfusion-fixed tumor xenograft sections were subjected to HGF-dependent glioma models alters HGF-induced cell sur- quantitative IR immunofluorescence by simultaneously vival, migration, and Akt activation. Our findings show that staining with primary antibodies specific for Cyr61 and Cyr61 induction contributes to HGF-dependent glioma cell glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Akt activation, migration, cell proliferation, and tumor xeno- methods described by Kearns and colleagues (ref. 32; graft growth. http://www.licor.com). Secondary antibodies labeled with two spectrally distinct near-IR dyes (IRDye 800 CW goat Materials and Methods anti-mouse, 1:10,000; IRDye 680 CW goat anti-rabbit, 1:10,000) were used to simultaneously detect and quantify Cyr61 rela- Cell culture and reagents. U373MG, U87MG, and SNB-19 tive to GAPDH. Computer-assisted signal quantification was cell lines were originally obtained from the American Type performed using the Odyssey Infrared Imager from LI-COR Culture Collection. Serial routine testing in our laboratory Biosciences. shows that these cells have maintained their characteristic The Johns Hopkins University Institutional Animal Care phenotypic and gene expression patterns. U87MG cells were and Use Committee approved all animal protocols used in grown in MEM with Earle salts and L-glutamine (MEM 1X; this study. Mediatech, Inc.) supplemented with 10% fetal bovine serum Immunohistochemistry and immunofluorescence. Cryo- (FBS; Gemini Bioproducts, Inc.), 2 mmol/L sodium pyruvate stat or paraffin-embedded sections were stained with anti- (Mediatech), 0.1 mmol/L MEM nonessential amino acids Cyr61, anti-total Met, or anti-Ki67 using previously described (Mediatech), and penicillin-streptomycin (Mediatech). methods (29). Biotinylated-conjugated secondary antibodies U373MG and SNB-19 cells were grown in DMEM low glucose followed by incubation with 3,3′-diaminobenzidine peroxi- with L-glutamine and sodium pyruvate supplemented with dase substrate were used to detect primary antibodies. Sec- 10% FBS and penicillin-streptomycin as previously described tions were counterstained with Gill's hematoxylin solution. (29). All cells were grown at 37°C in a humidified incubator We analyzed three to four random fields per histologic sec- with 5% CO2. tion and two sections per tumor to generate an average value Antibodies. Antibodies were obtained from the following
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