Research Article Thymosin hh10 Inhibits Angiogenesis and Tumor Growth by Interfering with Ras Function Seung-Hoon Lee,1 Myung Jin Son,1,2 Sun-Hee Oh,1 Seung-Bae Rho,1 Kyungsook Park,1 Yung-Jin Kim,2 Mi-Sun Park,1 and Je-Ho Lee1 1Molecular Therapy Research Center, Samsung Medical Center, School of Medicine, Sung Kyun Kwan University, Seoul, Korea and 2Department of Molecular Biology, Pusan National University, Busan, Korea Abstract are a family of highly conserved small peptides that inhibit barbed end actin polymerization by sequestering actin mono- Thymosin h10 is a monomeric actin sequestering protein mers (8). Among them, thymosin h4 and thymosin h10 are the that regulates actin dynamics. Previously, we and others h have shown that thymosin h acts as an actin-mediated two most abundant -thymosins in the mammalian species and 10 coexist in some tissue types at varying ratios (9). Although both tumor suppressor. In this study, we show that thymosin h10 is not only a cytoskeletal regulator, but that it also acts as a peptides share a high degree of sequence homology, they show potent inhibitor of angiogenesis and tumor growth by its distinct patterns of expression in several tissues (10) and play interaction with Ras. We found that overexpressed thymosin different roles during rodent development (11). Recently, the angiogenic effects of several members of the thymosin family of h10 significantly inhibited vascular endothelial growth factor–induced endothelial cell proliferation, migration, peptides were studied in the chick chorioallantoic membrane h a invasion, and tube formation in vitro. Vessel sprouting was model (12). Thymosin 4, prothymosin, and thymosin 1 were associated with enhancement of angiogenesis, whereas para- also inhibited ex vivo. We further show that thymosin h10 thymosin, thymosin h9, and thymosin h10 were associated with directly interacted with Ras. This interaction resulted in h inhibition of the Ras downstream mitogen-activated protein inhibition of angiogenesis. Thymosin 4 also stimulated tumor kinase/extracellular signal-regulated kinase kinase signaling metastasis by activating cell migration and angiogenesis (13, 14). Here, we did cDNA chip analysis to identify genes regulated pathway, leading to decreased vascular endothelial growth h factor production. Thymosin h injected into a xenograft by thymosin 10. The expression of genes related to 10 angiogenesis, cell migration, and proliferation was dramatically model of human ovarian cancer in nude mice markedly h inhibited tumor growth and reduced tumor vascularity. In inhibited by thymosin 10 in ovarian cancer cells, including Rac1 (15), nitric-oxide synthase (16), focal adhesion kinase (17), contrast, a related thymosin family member, thymosin h4, did not bind to Ras and showed positive effects on Lim kinase (LIMK1; ref. 18), Wave (WASF1; ref. 19), hypoxia- a angiogenesis. These findings show that the inhibition of inducible factor-1 (20), platelet-derived growth factor receptor h (21), ELK1 (22), and ARHGEF (23). From this data, it seems Ras signal transduction by thymosin 10 results in anti- h angiogenic and antitumor effects, suggesting that thymosin that thymosin 10 is involved in the inhibition of angiogenesis and tumor growth, although the underlying mechanisms are not h10 may be valuable in anticancer therapy. (Cancer Res 2005; 65(1): 137-47) fully understood. Using an adenovirus vector expressing thymosin h10, we found that thymosin h10 significantly inhibited VEGF-induced angiogenesis and tumor growth in vitro, ex vivo, Introduction and in vivo. These effects were mediated by thymosin h10 Angiogenesis, the sprouting of new capillaries from preexisting directly binding to Ras and interfering with its downstream vasculature, is an essential process in tumor growth (1, 2). Thus, signaling pathways. Therefore, thymosin h10 is a multifunctional angiogenesis-based therapies have become a very promising protein that inhibits Ras and its signaling pathways. These modality in the treatment of cancer (3). One of the key interactions regulate potent antiangiogenic and antitumor mediators of angiogenesis is the vascular endothelial growth effects. factor (VEGF), which can promote the survival, proliferation, and migration of endothelial cells (4). VEGF expression and secretion are stimulated in tumor cells by activation of oncogenes such as Materials and Methods Ras (5). VEGF-mediated Ras signaling in endothelial cells is also Cell Culture. Human umbilical vein endothelial cell (HUVEC; essential in angiogenic responses (6). Clonetics, San Diego, CA) was grown on 0.3% gelatin (Sigma, St. Louis, Previously, we reported that thymosin h10 was down-regulated MO) coated dishes using the EGM-2 Kit (Clonetics). Human ovarian in human ovarian cancer tissues (7). When thymosin h10 was cancer cells (2774) and 293 cells were cultured in DMEM and EMEM, overexpressed in ovarian cancer cells, it acted as a tumor respectively, and were supplemented with 10% fetal bovine serum (FBS) suppressor by disrupting the actin structure. The h-thymosins and antibiotics (Life Technologies, Gaithersburg, MD). Animals. Specific pathogen-free BALB/c and nu/nu mice were supplied by Biogenomics (Seoul, Korea) and Charles River Labs (Wilmington, MA), respectively. All animal studies were approved by the Animal Care and Use Note: S-H. Lee and M. J. Son contributed equally to this work. Committee of Samsung Medical Center. Requests for reprints: Je-Ho Lee or Seung-Hoon Lee, Molecular Therapy Research Adenovirus and Vector Construction. The construction of an Center, Samsung Medical Center, Annex 8F, 50 Ilwondong, Kangnamgu, Seoul, Korea. adenovirus vector for green fluorescence protein-thymosin h10 (GFP- Phone: 82-2-3410-6833; Fax: 82-2-3410-6829; E-mail: [email protected]; h [email protected]. AdT 10) was done as described previously (7). For the adenovirus vector D2005 American Association for Cancer Research. for thymosin h10 (AdTh10) construct, PCR-amplified full-length human www.aacrjournals.org 137 Cancer Res 2005; 65: (1). January 1, 2005 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Cancer Research thymosin h10 fragment was cloned into a HindIII/XhoIsiteof Ex vivo Angiogenesis Assay. A novel ex vivo angiogenesis assay using pDACMVp(A) vector. The adenoviruses were used at 100 multiplicity of explant culture of mouse skeletal muscle on Matrigel was done with infection for infection experiments. To construct pcDNA3.1-thymosin h4, some modifications, according to Jang et al. (25). Six-week-old BALB/c pcDNA3.1-thymosin h10, and pcDNA4HisMax-thymosin h4, PCR-amplified mice were anesthetized and the legs were shaved. The tibialis anterior full-length human thymosin h4 and thymosin h10 fragments were cloned muscle was extracted and then cross-sections of muscle were washed into the EcoRI/XhoI site of the pcDNA3.1 vector (Invitrogen) and of the thrice with PBS. The washed muscle was placed in a 24-well plate pcDNA4HisMax vector (Invitrogen), respectively. containing 200 AL of growth factor-reduced Matrigel and polymerized for Small Interfering RNA Construction and Transfection. The siRNA 30 minutes at 37C. M199 containing 1% FBS with or without 10 ng/mL oligonucleotide sequence targeting thymosin h10 (AAGCGGAGU- of VEGF was added. After 6 days, outgrowth of capillary-like structures GAAAUUUCCUAA) corresponded to nucleotides 199 to 217 in the human was observed and then fresh medium containing either 2  108 plaque- sequence. Small interfering RNA (siRNA) was synthesized by using a siRNA forming unit (pfu) of adenovirus or 10 nmol/L of paclitaxel was added. construction kit (Ambion, Austin, TX) and transfected by using the RNAi Media were changed every other day. After an additional 5 days, the shuttle (Orbigen, San Diego, CA) according to the manufacturer’s mean area of microvessels was measured by an optical imaging protocols. HUVECs were then infected with either GFP-AdTh10 alone or technique and quantified using ImageLab imaging software. Independent GFP-AdTh10 with siRNA transfection. GFP images were captured using a experiments were repeated thrice and each value represents the mean fluorescence microscope (Zeiss, Oberoken, Germany). Total RNA was F SD of triplicate samples. isolated with TRIZOL Reagent (Life Technologies) and reverse transcrip- Yeast Two Hybrid Analysis. LexA-human thymosin h10 or thymosin tion-PCR was done. h4 fusion protein was constructed and used to screen binding proteins [3H]Methylthymidine Incorporation Assay. To measure cell prolifer- from a human ovary cDNA library (Clontech, Palo Alto, CA). The binding ation, HUVECs were infected with either empty adenovirus, AdTh10,or proteins were expressed as B42 fusion proteins. cDNA encoding full AdTh10 + siRNA. To determine the effect of thymosin h4, HUVECs were length human K-Ras or H-Ras were PCR amplified and ligated separately transfected with either pcDNA3.1 or pcDNA3.1-thymosin h4 using the into the EcoRI/XhoI sites of the B42. Positive interactions were confirmed FuGENE 6 reagent (Roche, Mannheim, Germany). After 18 hours, cells by cell growth on leucine-depleted yeast synthetic medium and blue were incubated for 6 hours in M199 containing 1% FBS and then colony formation on 5-bromo-4-chloro-3-indolyl-h-D-galactoside (X-gal, stimulated with VEGF (10 ng/mL, R&D Systems, Minneapolis, MN) for 5 mmol/L)-containing medium. The activity of the interaction between 3 24 hours in M199 containing 1% FBS. [ H]methylthymidine (0.5 ACi/mL, thymosin h10 or thymosin h4 and Ras was determined by measuring the Amersham, Arlington Heights, IL) was added 4 hours prior to the assay. relative expression level of h-galactosidase. The h-galactosidase activity The cpm values from cultures were counted with a liquid scintillation was calculated using the formula units = [1,000  (A420 À 1.75  A550)]/ counter (Beckman, Fullerton, CA). Independent experiments were (time  volume  A600). repeated thrice and each value represents the mean F SD of triplicate Glutathione S-transferase Pull-Down Assay and Coimmunopreci- samples. pitation. Glutathione S-transferase–fused thymosin h10 and His-fused Migration and Invasion Assay.