Thymosin Hh10 Inhibits Angiogenesis and Tumor Growth by Interfering with Ras Function

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

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 Â 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 Â (A420 À 1.75 Â A550)]/ counter (Beckman, Fullerton, CA). Independent experiments were (time Â volume Â 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. Migration and invasion were assayed K-Ras were purified on a glutathione Sepharose 4B (Amersham using Transwells (8-Am pore size, Costar, Cambridge, MA) as described Pharmacia Biotech, Piscataway, NJ) and on a Ni-NTA Agarose (Qiagen, previously (24). For the migration assay, the lower surface of filter was Chatsworth, CA) according to the manufacturers’ instructions, respec- coated with 10-Am of gelatin. M199 containing 1% FBS with VEGF tively. Equal amounts of glutathione S-transferase or glutathione S-

(25 ng/mL) was placed in the lower wells. Uninfected, Ad-, AdTh10-, or transferase-thymosin h10 immobilized on glutathione Sepharose beads AdTh10 + siRNA–infected HUVECs at a final concentration of 1 Â were incubated with His-K-Ras. Coimmunoprecipitated K-Ras was 104 cells/100 AL were seeded into each of the upper wells and incubated detected by Western blot with anti-His antibody. For coimmunoprecipi- for 24 hours. Cells were fixed and stained with H&E. Nonmigrating cells tation in vivo, HUVECs were transiently transfected with GFP or GFP- on the upper surface of the filter were removed by wiping with a cotton Th10 usingtheFuGENE6reagent.TheendogenousRaswas swab. The number of cells that migrated to the lower side of the filter immunoprecipitated with anti-Ras antibody (Oncogene, Uniondale, NY) was counted under a light microscope and mean values of eight fields and coimmunoprecipitated thymosin h10 was detected by Western blot were determined. For the invasion assay, the lower surface and upper with an anti-GFP antibody. For coimmunoprecipitation with Ras and surface of filter was coated with 10 Ag of gelatin and 10 Ag of Matrigel thymosin h4, 2774 cells were transiently transfected with GFP-thymosin (BD Biosciences, Bedford, MA), respectively. Uninfected, Ad-, AdTh10-, or h10 or His-thymosin h4 using the FuGENE 6 reagent. The thymosin h10 AdTh10 + siRNA–infected HUVECs at a final concentration of 1 Â or thymosin h4 was immunoprecipitated with anti-GFP antibody or anti- 104 cells/100 AL in M199 containing 1% FBS with VEGF (25 ng/mL) were His antibody, respectively. The coimmunoprecipitated Ras was detected seeded into each of the upper wells and incubated for 30 hours. The by Western blot with an anti-Ras antibody. fixation and quantification methods are the same as that of the Labeling of the Actin Cytoskeleton. HUVECs were transfected with migration assay. To determine the effect of thymosin h4, HUVECs were either pcDNA3.1, pcDNA3.1-thymosin h4, pcDNA3.1-thymosin h10,or transfected with either pcDNA3.1 or pcDNA3.1-thymosin h4 using the pcDNA3.1-thymosin h4 with pcDNA3.1-thymosin h10 (1:1) using the FuGENE 6 reagent. Independent experiments were repeated thrice and FuGENE 6 reagent. After 72 hours, the cells were incubated for 2 hours each value represents the mean F SD of triplicate samples. in M199 containing 1% FBS and then stimulated with or without 50 ng/mL Tube Formation Assay. Growth factor–reduced Matrigel (200 ALof of VEGF for 15 minutes. Cells were fixed and stained with Alexa fluor 488 10 mg/mL) was added into a 24-well plate and polymerized for phalloidin (Molecular Probes, Eugene, OR). Images were analyzed using a

30 minutes at 37jC. Uninfected, Ad-, GFP-AdTh10–, or GFP-AdTh10 + fluorescence microscope with a digital CCD camera (Olympus, Lake siRNA–infected HUVECs (1 Â 105 cells) were seeded on the surface of Success, NY). the Matrigel. Cells were then incubated for 48 hours with or without Ras activation Assay. Uninfected, Ad-, or AdTh10-infected HUVECs were 10 ng/mL of VEGF in M199 containing 1% FBS. Morphologic changes of serum-deprived overnight and stimulated with or without VEGF (50 ng/mL) the cells were photographed at Â40 magnification. HUVEC tube length for 5 minutes in M199 containing 1% FBS (6). The cell lysate was incubated was determined using an inverted microscope with a digital CCD camera with glutathione S-transferase-Raf1-Ras-binding domain (RBD) in the (Zeiss) and quantified using ImageLab imaging software (MCM Design). presence of an immobilized Glutathione Disc (Pierce). The assay was done To determine the combined effect of thymosin h4 and thymosin h10, according to the manufacturers’ instructions. The pull-down active Ras was HUVECs were transfected with either pcDNA3.1, pcDNA3.1-thymosin h4, detected by Western blot analysis using anti-Ras antibody. or pcDNA3.1-thymosin h10 using the FuGENE 6 reagent. Independent Ras Guanidine Nucleotide Binding Assay. The assay was done as experiments were repeated thrice and each value represents the mean described (26) with minor modifications. Uninfected, Ad-, or AdTh10 F SD of triplicate samples. infected HUVECs were serum-deprived overnight, labeled with 0.2 mCi/

Cancer Res 2005; 65: (1). January 1, 2005 138 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Thymosin hhh10 Inhibits Angiogenesis mL of [32P] orthophosphate (Amersham) for 3 hours in a phosphate-free GFP-2774 cells were prepared by stable transfection of pEGFP-C1 medium (Life Technologies), and stimulated with or without VEGF (Clontech). GFP-expressing tumors were examined using the Illumatool (50 ng/mL) for 5 minutes. Cell lysates were harvested and Ras proteins tunable lighting system (Lightools Research, Encinitas, CA). An intra- 9 were immunoprecipitated with anti-Ras antibody. Bound guanine tumoral injection of 1 Â 10 pfu/20 AL of AdTh10 was done on day 7 nucleotides were eluted from precipitated protein complexes and after tumor injection. Mice were sacrificed on day 10 after virus analyzed by TLC using polyethyleneimine-cellulose plates (Sigma). The injection. Tumors were then excised and prepared for immunohisto- presence of Ras GDP and GTP was assessed by autoradiography and the chemistry. Frozen sections were stained with rat monoclonal anti-mouse ratio of GTP to GTP + GDP was determined by densitometry. Similar CD31 (PECAM-1) antibody (PharMingen, San Diego, CA). Vascular density results were obtained in three independent experiments. in the tumors was calculated by counting the number of blood vessels in Subcellular Fractionation of Cell Lysates, Western Blot, and three separate tumor cross-sections per group. The specificity of the

Immunoprecipitation. Uninfected, Ad-, or GFP-AdTh10–infected staining was confirmed with isotype-matched antibodies (normal rat HUVECs were stimulated with or without VEGF (50 ng/mL) for 5 minutes IgG1n). and separated into cytosol, membrane, and nuclear fractions according to Data Analysis and Statistics. Values are presented as the mean F SD the manufacturer’s protocols (Calbiochem, La Jolla, CA). Fractionated or or F SE. Statistical comparisons between groups were done using the total proteins were immunoblotted with specific antibodies to GFP, pMEK, Student’s t test. P < 0.05 was considered statistically significant. pERK, extracellular signal-regulated kinase (ERK), and VEGF (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA), as well as tubulin antibody (Innogenex, San Ramon, CA). For VEGF immunoprecipitation, concentrated 2774 cell conditioned medium (27) was incubated with Results

VEGF antibody as previously described (28). Thymosin h10 Inhibits VEGF-Induced Proliferation, Migra- S.C. and Orthotopic Tumor Models and Immunohistochemistry. To 6 tion, and Invasion of HUVECs. To determine the effects of establish tumors in mice, 1 Â 10 of 2774 tumor cells were injected s.c. thymosin h on endothelial cell functions crucial to angiogenesis, in the mid-dorsal region. Tumors were allowed to grow for 14 days. 10 9 its effects on VEGF-induced proliferation, migration and invasion Then, an intratumor al injection of 1 Â 10 pfu/40 AL of AdTh10 was done thrice, once every 3 days. Tumor size was evaluated by caliper of endothelial cells were investigated (Fig. 1). HUVECs were either h h measurements every 3 days. Mice were sacrificed on day 27 after final uninfected or infected with Ad, AdT 10, or AdT 10 with siRNA h virus injection. Tumors were then excised and prepared for immunohis- (thymosin 10–targeted small interfering RNA), then DNA synthesis 3 tochemistry. For the orthotopic model of 2774 tumor growth, 1 Â 106 of was assayed using [ H]thymidine incorporation (Fig. 1A). As GFP-2774 cells were injected into the right ovary through the fat pad. expected, VEGF increased DNA synthesis of uninfected HUVECs

Figure 1. Thymosin h10 inhibits endothelial cell proliferation, migration, and invasion in vitro. A, HUVECs were either uninfected (Un) or infected with Ad, AdTh10, or AdTh10 with siRNA transfection (AdTh10 + siRNA) for 18 hours followed by treatment with or without VEGF (10 ng/mL) for 24 hours. cpm value of [3H]thymidine was determined with a liquid scintillation counter. B, ablation of GFP-Th10 protein and mRNA expression by siRNA in HUVECs. Bar, 50 Am. C and D, uninfected HUVECs and adenovirus-infected HUVECs were seeded on Transwells for the migration assays (C ) or on Matrigel-coated Transwells for the invasion assays (D) followed by stimulation with or without VEGF (25 ng/mL) for 24 or 30 hours, respectively. Number of migrated or invaded cells was counted under a light microscope and mean values were determined. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with VEGF alone.

www.aacrjournals.org 139 Cancer Res 2005; 65: (1). January 1, 2005

and of empty virus-infected HUVECs when compared with Fig. 3C). These results indicate that thymosin h10 directly interacts unstimulated cells (29). Overexpression of thymosin h10 signifi- with Ras. cantly inhibited VEGF-induced DNA synthesis. This inhibitory Thymosin h4 neither Interacts with Ras nor Inhibits effect was not due to cytotoxicity of thymosin h10 in endothelial Angiogenesis. To establish the functional consequence of the cells, since thymosin h10 had no effect on the viability of HUVECs binding of thymosin h10 to Ras, we compared the Ras binding in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ability and the effects on angiogenesis of thymosin h4 with those assay (data not shown). In addition, thymosin h10 did not have of thymosin h10, since these proteins apparently share some cytotoxic effects on other normal cell types tested, such as normal structural features and functionalities (8, 9), but have divergent fibroblasts, MRC-5 and IMR-90 (data not shown). To further effects on angiogenesis (12, 14). First, we determined whether confirm the effect of thymosin h10 on endothelial proliferation, we thymosin h4 also binds to Ras. Interestingly, thymosin h4 did not used RNA interference (30). As shown in Fig. 1B, siRNA markedly bind to Ras in vivo (Fig. 4A and B). Furthermore, consistent with inhibited the expression of GFP-thymosin h10 protein and mRNA in other reports (14, 31), thymosin h4 had no effect on proliferation HUVECs. However, siRNA did not affect the expression of an and invasion of HUVECs (Fig. 4C and D), whereas thymosin h10 irrelevant gene (GAPDH). The inhibitory effect of thymosin h10 on showed inhibitory effects on the activities (Fig. 1). In addition, VEGF-induced endothelial cell proliferation was completely thymosin h4 increased migration and tube formation of HUVECs restored by siRNA transfection. The results indicate that VEGF- (Fig. 4E and F) in contrast to thymosin h10 which was inhibitory induced endothelial cell proliferation is specifically inhibited by (Figs. 1 and 2A). To investigate the combined effect of thymosin thymosin h10. h4 and thymosin h10 on the tube formation, both thymosins were To investigate whether overexpressed thymosin h10 modulates simultaneously overexpressed (Fig. 4G). The positive effect of the effects of VEGF on endothelial cell migration and invasion, we thymosin h4 on tube formation was overridden by increasing did Transwell migration and invasion assays. VEGF enhanced the concentrations of thymosin h10, and vice versa. However, migration (Fig. 1C) and invasion (Fig. 1D) of uninfected HUVECs thymosin h4 and thymosin h10 disrupted VEGF-induced stress and of empty virus-infected HUVECs when compared with that of fiber formation of HUVECs at a similar degree (Fig. 4H). In unstimulated cells as expected. However, overexpression of addition, simultaneous overexpression significantly inhibited the thymosin h10 significantly reduced VEGF-induced migration and reorganization of the actin architecture in response to VEGF. invasion of HUVECs. The ablation of overexpressed thymosin h10 Therefore, the effects of thymosin h10 when compared with that by siRNA maintained the stimulatory effects of VEGF on of thymosin h4, suggest that inhibition of angiogenesis by migration and invasion of HUVECs. Therefore, overexpressed thymosin h10 is via a direct interaction with Ras, independent thymosin h10 potently inhibited key events of the angiogenic of actin binding. process induced by VEGF, such as proliferation, migration, and Thymosin h10 Interferes With Ras Function. To understand invasion of endothelial cells in vitro. the role of thymosin h10 binding to Ras, we first investigated Thymosin h10 Inhibits Tube Formation In vitro and Vessel whether thymosin h10 is involved in Ras activity. The stimulation Sprouting Ex vivo. To confirm that thymosin h10 has direct of Ras and subsequent activation of its downstream effector antiangiogenic effects, we investigated whether overexpression of molecules is crucial in angiogenesis (6, 32). Based on the high thymosin h10 could alter endothelial tube formation. Uninfected affinity of active Ras (Ras-GTP) for the RBD of Raf, a downstream or empty virus-infected cells incubated with VEGF formed an effector of activated Ras (33), Ras activation was assayed. organized network of endothelial cells on Matrigel (Fig. 2A). In Stimulation of serum-deprived HUVECs with VEGF resulted in contrast, overexpression of thymosin h10 markedly inhibited a robust increase of Raf bound Ras-GTP. On the other hand, VEGF-induced tube formation. The inhibitory effect of thymosin overexpression of thymosin h10 in these cells resulted in the h10 on VEGF-induced tube formation was completely restored by absence of the active Ras (Fig. 5A). siRNA transfection. We next characterized the molecular mechanism by which To evaluate whether the thymosin h10 inhibits vessel sprouting, thymosin h10 inhibits Ras function. To determine whether an ex vivo explant assay (25) was done (Fig. 2B). Abundant vessel thymosin h10 decreases the amount of Ras-GTP or interferes sprouting was detected in uninfected or empty virus-infected with Raf binding of Ras-GTP, we did a Ras guanidine nucleotide explants in the presence of VEGF. In contrast, overexpression of binding assay (Fig. 5B). Very little GTP was detected in untreated thymosin h10 showed significantly reduced VEGF-induced vessel cells, but significant amounts of GTP were found in cells treated sprouting at levels lower than paclitaxel, a cytotoxic cancer drug with VEGF. Contrary to our expectation, thymosin h10 markedly with antiangiogenic activities. These observations suggest that increased the level of GTP. Therefore, it is possible that thymosin thymosin h10 effectively suppressed capillary formation in vitro h10 interferes with Raf binding of Ras-GTP rather than decreasing and ex vivo. the amount of Ras-GTP. This may result in the inhibition of the Thymosin h10 Interacts With Ras. We next tried to determine GTPase activity of Ras and the subsequent accumulation of Ras- the mechanism involved in the inhibition of angiogenesis by GTP. The subcellular localization of thymosin h10 supports this identifying proteins which bind to thymosin h10. Thus, we screened notion (Fig. 5C). GFP-Th10 was not only detected in the cytosolic for thymosin h10 binding proteins using the yeast two-hybrid fraction, but also in the membrane fraction (top), indicating that system. One prominent gene identified was Ras. Positive interaction thymosin h10 may bind Ras in the membrane and interfere with was verified by both cell growth and the h-galactosidase assay Ras signaling. However, thymosin h10 did not affect Ras (Fig. 3A). Direct interaction of Ras with thymosin h10 in vitro localization (middle). was confirmed using a glutathione S-transferase pull-down assay Thymosin h10 Inhibits ERK Signaling in Endothelial Cells (Fig. 3B). The interaction of the two proteins was also shown by and Reduces VEGF Expression in Both Endothelial and Tumor co-immunoprecipitation between the endogenous Ras and the Cells. Based on the above findings, we investigated whether exogenously introduced GFP-tagged thymosin h10 (GFP-Th10; thymosin h10 inhibits Ras downstream mitogen-activated protein

Cancer Res 2005; 65: (1). January 1, 2005 140 www.aacrjournals.org

Figure 2. Thymosin h10 inhibits tube formation in vitro and vessel sprouting ex vivo. A, uninfected HUVECs and indicated adenovirus-infected HUVECs were plated on growth factor-reduced Matrigel and treated with or without VEGF (10 ng/mL) for 48 hours. The formation of tubular structures was detected by an inverted microscope. Arrows, thin and broken tubes. GFP Images were captured using a fluorescence microscope. Bar, 100 Am. Tube length was quantified and expressed as the means F SD. *, P < 0.05; ***, P < 0.001 compared with VEGF alone. B, cross-sections of mouse tibialis anterior muscle were embedded in growth factor–reduced Matrigel with or without VEGF for 6 days and treated with Ad, AdTh10, or paclitaxel for 5 days. Outgrowth of capillary-like structures was observed with an inverted microscope (magnifications, Â12.5 and Â40, top and bottom, respectively). Bar, 500 Am. Mean area of vascular sprouting was quantified and expressed as the means F SD. **, P < 0.01 compared with VEGF alone. www.aacrjournals.org 141 Cancer Res 2005; 65: (1). January 1, 2005

Figure 3. Thymosin h10 interacts with Ras in vitro and in vivo. A, transformants were assayed for their ability to grow on medium lacking leucine (left) and for h-galactoside expression (right ). Activity of the interaction between thymosin h10 and Ras was represented by the relative activity of h-galactosidase expression. B, the GST pull-down assay was done using an anti-GST antibody. K-Ras was detected with an anti-His antibody. C, HUVECs were transiently transfected with GFP or GFP-Th10. Total cell lysates were immunoprecipitated with an anti-Ras antibody. The presence of thymosin h10 in the immunoprecipitates was detected using an anti-GFP antibody. The immunoprecipitates and total cell extracts were analyzed by Western blots with an anti-Ras antibody and anti-GFP antibody, respectively.

kinase kinase (MEK) and ERK activation (Fig. 5D). Ras-ERK volume of thymosin h10–treated tumors was 77% smaller than mediated transcriptional up-regulation of angiogenic factors, such those from control mice (Fig. 6C). Moreover, immunohistologic as VEGF, is a well-known mechanism that promotes angiogenesis staining of endothelial cells in the thymosin h10–treated mice (29). VEGF-stimulated MEK and ERK phosphorylation were showed an 89% decrease in the number of blood vessels stained markedly reduced by overexpressed thymosin h10. However, the with CD 31. total ERK level was unaffected by thymosin h10. Therefore, it seems These antitumor and antiangiogenic effects of thymosin h10 that thymosin h10 inhibits the Ras-ERK signaling pathway in were confirmed in orthotopically injected tumor cells (Fig. 6D). We HUVECs. Consistent with these findings, overexpressed thymosin injected GFP-2774 tumor cells into the right ovary and did h10 completely inhibited VEGF expression in HUVECs (Fig. 5E). This intratumoral injections of thymosin h10 on day 7 after tumor suggests that thymosin h10 inhibits the autocrine effect of VEGF in injection. GFP-expressing tumors were significantly decreased in endothelial cells and thus, has a direct antiangiogenic effect. thymosin h10–treated mice. The volume of excised tumors on day Next, we investigated the effect of thymosin h10 on VEGF 10 after virus injection was 54% smaller than those from control production in tumor cells. VEGF is mainly secreted by tumor cells mice. Also, the erythema of the tumor due to induction of to recruit VEGF receptor–expressing endothelial cells to the angiogenesis was dramatically reduced in thymosin h10–treated tumor (29). Overexpression of thymosin h10 in 2774 ovarian mice when compared with control mice. Immunohistologic cancer cells resulted in a marked reduction of VEGF expression staining of endothelial cells in thymosin h10–treated tumors and secretion into the medium (Fig. 5F). Thus, thymosin h10 showed a 77% decrease in the number of blood vessels. Together decreases VEGF production in tumor cells leading to a sup- these results show that overexpressed thymosin h10 potently pressed paracrine effect of VEGF on angiogenesis. suppresses angiogenesis and tumor growth in vivo. Thymosin h10 Inhibits Tumor Growth and Associated Angiogenesis. To explore whether thymosin h10 has direct antitumor activity, we tested the effects of overexpressed Discussion thymosin h10 on tumor cell growth in vitro. We found that thymosin h10 markedly decreased 2774 ovarian cancer cell growth This study shows the crucial function of a monomeric actin- when compared with controls (Fig. 6A). sequestering protein, thymosin h10, as a new potent antiangiogenic The antitumor and antiangiogenic activity of thymosin h10 was and antitumor molecule that targets Ras. Here, we suggest a then evaluated in vivo. Cells (2774) were implanted s.c. in nude possible mechanism for the inhibition of angiogenesis and tumor mice. We allowed the tumors to grow until they reached a mean growth by thymosin h10, which involves direct binding to Ras volume of 100 mm3. On day 14, an intratumoral injection of thereby inhibiting the Ras-activated MEK/ERK signaling pathway. 9 1 Â 10 pfu/40 AL of thymosin h10 was done and repeated every Ras effector pathways not only affect tumor cell proliferation and 3 days for 9 days. Tumor growth was significantly inhibited by survival (34) but also lead to the induction of angiogenesis (5, 6). thymosin h10 (Fig. 6B) with no signs of body weight loss or liver Induction occurs mainly by means of ERK-mediated transcriptional toxicity (data not shown). Tumors from thymosin h10–treated up-regulation of angiogenic factors and increased invasiveness mice were excised on day 27 after the final virus injection. The through both ERK-mediated expression of matrix metalloprotei-

Cancer Res 2005; 65: (1). January 1, 2005 142 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Thymosin hhh10 Inhibits Angiogenesis nases and Rac-mediated effects on the cytoskeleton (35). Therefore, result in a synergistic effect greater than that of blocking any one Ras-targeted thymosin h10 could play a key role in inhibiting tumor cellular response alone. This could be explained by the fact that Ras growth and angiogenesis. proteins operate as molecular switches in signaling pathways that In this study, overexpressed thymosin h10 potently inhibited regulate diverse cell growth and differentiation processes (34, 36). multiple angiogenic processes, including endothelial cell prolifera- Ras proteins are involved in intracellular signaling from receptor tion, migration, invasion, tube formation, and vessel sprouting tyrosine kinases which results in the activation of a phosphorylation (Figs. 1 and 2). Cell viability was not affected by empty adenoviruses cascade (37). Growth factors, such as VEGF, fibroblast growth factor, or by thymosin h10–expressing adeonovirus in the angiogenesis platelet-derived growth factor, nerve growth factor, epidermal assays (data not shown). The combined inhibitory effects of growth factor, and insulin activate Ras proteins, but in some cases h thymosin h10 on critical endothelial functions in angiogenesis may other factors, such as transforming growth factor- (38) and

Figure 4. Thymosin h4 neither interacts with Ras nor inhibits angiogenesis. A, transformants were assayed for their ability to grow on medium lacking leucine (left ) and for h-galactoside expression (right ). The activity of the interaction between thymosin h4 and Ras was represented by the relative activity of h-galactosidase expression. B, 2774 cells were transiently transfected with GFP-thymosin h10 and His-thymosin h4. thymosinh10 or thymosin h4 was immunoprecipitated with anti-GFP antibody or anti-His antibody, respectively. The presence of Ras in the immunoprecipitates and the endogenous Ras was detected using an anti-Ras antibody. C, 3 HUVECs were transfected with thymosin h4 for 18 hours followed by treatment with or without VEGF (10 ng/mL) for 24 hours. cpm value of [ H]thymidine was determined with a liquid scintillation counter. D, untransfected HUVECs and indicated vector transfected HUVECs were seeded on Matrigel-coated Transwells, followed by stimulation with or without VEGF (25 ng/mL) for 30 hours. Number of invaded cells was counted under a light microscope and mean values were determined. E, untransfected HUVECs and indicated vector transfected HUVECs were seeded on Transwells followed by stimulation with or without VEGF (25 ng/mL) for 24 hours. Number of migrated cells was counted under a light microscope and mean values were determined. F, untransfected HUVECs and the indicated vector transfected HUVECs were plated on growth factor-reduced Matrigel and then treated with or without VEGF (10 ng/mL) for 24 hours. Tube length was quantified and expressed as the means F SD. **, P < 0.01 compared with VEGF alone. G, HUVECs were transfected with thymosin h4 and thymosin h10, depending on the concentration of each plasmid and their ratio, and the tube formation assay was done. The effect on tube formation is represented as % change of control. H, HUVECs were transfected with thymosin h4 and thymosin h10 for 72 hours followed by stimulation with or without VEGF (50 ng/mL) for 15 minutes. Cells were fixed and stained with phalloidin (green) for filamentous actin. Images were captured on a fluorescence microscope. www.aacrjournals.org 143 Cancer Res 2005; 65: (1). January 1, 2005

Figure 5. Thymosin h10 interferes with VEGF-mediated Ras- ERK signaling in HUVECs and reduces VEGF production in HUVECs and in 2774 ovarian cancer cells. A, active Ras (Ras- GTP) in HUVECs was detected by using a GST-Raf1-RBD pull-down assay. Total Ras was detected by using anti-pan Ras antibody. B, thin layer chromatogram of the nucleotides eluted from immunoprecipitates of Ras from HUVECs. Ratio of GTP to GTP + GDP was determined by densitometry. C, cytosol, membrane, and nuclear extracts were prepared from uninfected 1, VEGF + Ad–infected 2 and VEGF + GFP-AdTh10–infected 3 HUVECs. Localization of GFP-Th10 and Ras was determined by Western blotting. Purity of the extracts was confirmed by Western blotting for tubulin. D, HUVECs were either uninfected or infected with Ad or GFP-AdTh10 for 48 hours followed by treatment with or without VEGF (50 ng/mL) for 15 minutes. Phosphorylated MEK and ERK and total ERK were detected by Western-blot analysis. E, the total cell lysate of HUVECs in D was used for VEGF detection. F, 2774 ovarian cancer cells were either uninfected or infected with Ad or AdTh10 for 48 hours. VEGF expression was detected by Western blot analysis (top). Secreted VEGF was detected in the concentrated conditioned medium (CCM) of 2774 by immunoprecipitation (bottom).

angiotensin-2 (39) activate Ras as well. Multiple downstream We had expected another possible mechanism by which h effectors have also been identified which may lead to alternate thymosin 10 inhibits angiogenesis via disruption of the actin pathways. Indeed, we found that thymosin h10 also inhibited Rac cytoskeleton of HUVECs, because depolymerization of actin stress activation by suppressing the mRNA expression of the guanosine fiber is well known function of h-thymosins (8, 9). However, 3 h h nucleotide exchange factor, vav (40). Therefore, when the function thymosin 4 and thymosin 10 showed the same inhibitory effect of the key regulator, Ras was blocked by thymosin h10 over- on actin polymerization, although they had opposite effects on expression, upstream signals from receptors for various factors and angiogenesis (Fig. 4). In addition, within the h-thymosin family, the downstream pathways were inhibited. These functions eventually actin binding motif (LKKTETQ) is highly conserved (41), and the lead to the suppression of angiogenesis. seven amino acids motif is essential for their angiogenic activity (42). Thus, angiogenesis inhibition by thymosin h10 is distinct from 3 Unpublished observations. the common actin binding property of h-thymosins. One possible

Cancer Res 2005; 65: (1). January 1, 2005 144 www.aacrjournals.org

Figure 6. Thymosin h10 inhibits tumor growth and angiogenesis in vivo. A, 2774 ovarian cancer cells were either uninfected or infected with Ad or AdTh10 for 48 hours. Number of cells was counted under the microscope. B, 2774 tumor cells were injected s.c. An intratumoral injection of Ad (5) and AdTh10 (.) was done thrice. Tumor volume is shown as the mean from 4 to 6 mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with Ad. C, tumor site and excised tumors from Ad- and AdTh10-treated mice on day 27 after virus injection were photographed. Tumor volume is shown as the mean from four mice (n = 8 per group). Frozen sections of the tumors were stained for endothelial cells using an anti-CD31 antibody. Bar, 50 Am. **, P < 0.01 compared with Ad. D, GFP-2774 cells were injected orthotopically into the right ovary. GFP expressing tumors (green) were examined using an UV illuminating system (top). The excised tumors (white arrows) and normal ovaries (white arrowheads) from Ad-and AdTh10-treated mice on day 10 after virus injection were photographed (middle). Tumor volume is shown as the mean from five mice. Frozen sections of the tumors were stained for endothelial cells using anti-CD31 antibody. Bar, 50 Am. *, P < 0.05 and **, P < 0.01 compared with Ad.

h h explanation is that thymosin h4 and thymosin h10 bind to G-actin On the other hand, thymosin 4 and thymosin 10 showed in a 1:1 complex forming a large pool of unpolymerized actin that distinct patterns of expression in several tissues (10) and played h can be easily released when needed for polymerization of actin different roles during rodent development (11). Thymosin 10 filaments. However, thymosin h10 inhibits multiple signaling mRNA levels were very low in the cardiovascular system of early h molecules needed for polymerization of actin filaments, such as mouse embryo, in contrast to thymosin 4 mRNA levels (43). Rac and Wave4 by interfering with Ras or directly, which results in Angiogenesis actively occurs in early development and is disrupting actin dynamics. This may explain why the two commonly controlled by the balance between angiogenic and homologous proteins have very different effects on angiogenesis. antiangiogenic factors depending on the demand of the physiologic environment in a development-dependent manner. These literature 4 Unpublished observations. findings further support the assumption that thymosin h4 and www.aacrjournals.org 145 Cancer Res 2005; 65: (1). January 1, 2005

thymosin h10 act on vessel development in a complementary way also be modified by geranylgeranyltransferase. The combined in vivo, and this may also be extended to the angiogenesis process. use of farnesyltransferase inhibitors and geranylgeranyltransfer- This hypothesis is also supported by the fact that overexpression ase inhibitors (44) has failed because they act on other proteins of thymosin aˆ4 and thymosin h10 induces an increased (14) and which are necessary for normal cell growth and show cellular decreased (Fig. 5E and F) expression of VEGF, respectively. toxicity. We now propose that thymosin h10 may overcome the h Thymosin 10 has direct effects on tumor cells. It inhibits 2774 deficiencies of these existing therapies that target Ras or its h ovarian cancer cell growth. In addition, we found that thymosin 10 signaling pathways. Because thymosin h10 directly binds to Ras inhibited Ras-ERK signaling (data not shown) as well as VEGF and interferes with Ras itself, it acts selectively in its specific secretion (Fig. 5) in these cells. Together, these observations suggest inhibition of Ras. Therefore, thymosin h10 could have a greater that overexpression of thymosin h10 in whole tumors could disrupt inhibitory effect on tumor cells and/or tumor vessels containing tumor growth and associated angiogenesis through both tumor cell- highly activated Ras compared with normal cells. mediated effects and effects on endothelial cells. This would be In conclusion, thymosin h10 is not only an actin-sequestering expected to be more potent than targeting either cell type alone. It protein. It has also been found to block the cellular signaling was also observed that thymosin h10 increased phospho-p53 in cascades involved in angiogenesis and in tumor growth. This newly ovarian cancer cells (data not shown) suggesting the possibility discovered mechanism may lead to the future development of h that thymosin h10 may be related to multiple effector pathways. effective cancer therapies using thymosin 10. Targeting the Ras proteins and their signaling pathways could be very valuable in developing cancer therapies (35). Over 20 cancer therapeutic agents have been developed thus far, but specific Acknowledgments inhibitors of upstream activators or downstream mediators showed limited effect on Ras activity. Prenylation, the post- Received 5/6/2004; revised 8/24/2004; accepted 10/22/2004. translational modification step, is required for the localization and Grant support: Korea Science and Engineering Foundation SRC (J-H. Lee) and National Institute of Toxicological Research (S-H. Lee). function of Ras. However, attempting to inhibit prenylation by The cost of publication of this article were defrayed in part by the payment of page using farnesyltransferase inhibitors has not been successful in charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. human trials. Although H-Ras is exclusively modified by We thank Drs. Hyun Seok Song and Seung Hee Hong for critically reviewing the farnesyltransferase, K-Ras and, to a lesser extent, N-Ras can manuscript.

References membrane angiogenesis model. Biochim Biophys Acta 23. Cherfils J, Chardin P. GEFs: structural basis for their 2001;1568:60–6. activation of small GTP-binding proteins. Trends 1. Risau W. Mechanism of angiogenesis. Nature 1997; 13. Malinda KM, Goldstein AL, Kleinman HK. Thy- Biochem Sci 1999;24:306–11. 386:671–4. mosin h 4 stimulates directional migration of 24. Lee OH, Kim YM, Lee YM, et al. Sphingosine 2. Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992; human umbilical vein endothelial cells. FASEB J 1-phosphate induces angiogenesis: its angiogenic action 267:10931–4. 1997;11:474–81. and signaling mechanism in human umbilical vein 3. Folkman J. Clinical applications of research on 14. Cha HJ, Jeong MJ, Kleinman HK. Role of thymosin h4 endothelial cells. Biochem Biophys Res Commun angiogeneisis. N Engl J Med 1995;333:1757–63. in tumor metastasis and angiogenesis. J Natl Cancer 1999;264:743–50. 4. Ferrara N. VEGF and the quest for tumour angiogen- Inst 2003;95:1674–80. 25. Jang HS, Kim HJ, Kim JM, et al. A novel ex vivo esis factors. Nat Rev Cancer 2002;2:795–803. 15. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, angiogenesis assay based on electroporation-mediated 5. Rak J, Mitsuhashi Y, Sheehan C, et al. Oncogenes and Hall A. The small GTP-binding protein rac regulates delivery of naked plasmid DNA to skeletal muscle. Mol tumor angiogenesis: differential modes of vascular growth factor-induced membrane ruffling. Cell 1992;70: Ther 2004;9:464–74. endothelial growth factor up-regulation in ras-trans- 401–10. 26. Downward J, Graves JD, Warne PH, Rayter S, Cantrell formed epithelial cells and fibroblasts. Cancer Res 16. Morbidelli L, Donnini S, Ziche M. Role of nitric oxide DA. Stimulation of p21ras upon T-cell activation. Nature 2000;60:490–8. in the modulation of angiogenesis. Curr Pharm Des 1990;346:719–23. 6. Meadows KN, Bryant P, Pumiglia K. Vascular endo- 2003;9:521–30. 27. Kim MS, Kwon HJ, Lee YM, et al. Histone deacetylases thelial growth factor induction of the angiogenic pheno- 17. Haskell H, Natarajan M, Hecker TP, et al. Focal induce angiogenesis by negative regulation of tumor type requires Ras activation. J Biol Chem 2001;276: adhesion kinase is expressed in the angiogenic blood suppressor genes. Nat Med 2001;7:437–43. 49289–98. vessels of malignant astrocytic tumors in vivo and 28. Song HS, Son MJ, Lee YM, et al. Oxygen tension 7. Lee SH, Zhang W, Choi JJ, et al. Overexpression of the promotes capillary tube formation of brain micro- regulates the maturation of the blood-brain barrier. thymosin h-10 gene in human ovarian cancer cells vascular endothelial cells. Clin Cancer Res 2003;9: Biochem Biophys Res Commun 2002;290:325–31. disrupts F-actin stress fiber and leads to apoptosis. 2157–65. 29. Plate KH, Breier G, Weich HA, Risau W. Vascular Oncogene 2001;20:6700–6. 18. Stanyon CA, Bernard O. LIM-kinase1. Int J Biochem endothelial growth factor is a potential tumour 8. Yu FX, Lin SC, Morrison-Bogorad M, Atkinson MA, Yin Cell Biol 1999;31:389–94. angiogenesis factor in human gliomas in vivo. Nature HL. Thymosin h10 and thymosin h4 are both actin 19. Yamazaki D, Suetsugu S, Miki H, et al. WAVE2 is 1992;29:845–8. monomer sequestering proteins. J Biol Chem 1993;268: required for directed cell migration and cardiovascular 30. Bosher JM, Labouesse M. RNA interference: genetic 502–9. development. Nature 2003;424:452–6. wand and genetic watchdog. Nat Cell Biol 2000;2: 9. Nachmias VT. Small actin-binding proteins: the h- 20. Ryan HE, Lo J, Johnson RS. HIF-1a is required for E31–6. thymosin family. Curr Opin Cell Biol 1993;5:56–62. solid tumor formation and embryonic vascularization. 31. Grant DS, Kinsella JL, Kibbey MC, et al. Matrigel 10. Lin SC, Morrison-Bogorad M. Developmental ex- EMBO J 1998;17:3005–15. induces thymosin h4 gene in differentiating endothelial pression of mRNAs encoding thymosins h4 and h10 21. Marx M, Perlmutter RA, Madri JA. Modulation of cells. J Cell Sci 1995;108:3685–94. in rat brain and other tissues. J Mol Neurosci 1990;2: platelet-derived growth factor receptor expression in 32. Eliceiri BP, Klemke R, Stromblad S, Cheresh DA. 35–44. microvascular endothelial cells during in vitro angio- Integrin avh3 requirement for sustained mitogen- 11. Anadon R, Rodriguez, Moldes I, et al. Differential genesis. J Clin Invest 1994;93:131–9. activated protein kinase activity during angiogenesis. expression of thymosins h (4) and h (10) during rat 22. Cheng JJ, Wung BS, Chao YJ, Wang DL. Sequential J Cell Biol 1998;140:1255–63. cerebellum postnatal development. Brain Res 2001;894: activation of protein kinase C (PKC)-a and PKC-q 33. Pumiglia KM, LeVine H, Haske T, Habib T, Jove R, 255–65. contributes to sustained Raf/ERK1/2 activation in Decker SJ. A direct interaction between G-protein h g 12. Koutrafouri V, Leondiadis L, Avgoustakis K, et al. endothelial cells under mechanical strain. J Biol Chem subunits and the Raf-1 protein kinase. J Biol Chem 1995; Effect of thymosin peptides on the chick chorioallantoic 2001;276:31368–75. 270:14251–4.

Cancer Res 2005; 65: (1). January 1, 2005 146 www.aacrjournals.org

34. Joneson T, Bar-Sagi D. Ras effectors and their role 38. Mulder KM, Morris SL. Activation of p21ras by multiple functions. Int J Biochem Cell Biol 2001;33: in mitogenesis and oncogenesis. J Mol Med 1997;75: transforming growth factor h in epithelial cells. J Biol 205–20. 587–93. Chem 1992;267:5029–31. 42. Philp D, Huff T, Gho YS, Hannappel E, Kleinman HK. 35. Downward J. Targeting RAS signalling pathways in 39. Sadoshima J, Izumo S. Signal transduction pathways The actin binding site on thymosin h4 promotes cancer therapy. Nat Rev Cancer 2003;3:11–22. of angiotensin II–induced c-fos gene expression in angiogenesis. FASEB J 2003;17:2103–5. 36. Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, cardiac myocytes in vitro. Roles of phospholipid-derived 43. Carpintero P, Franco del Amo F, Anadon R, Gomez- Der CJ. Increasing complexity of Ras signaling. Onco- second messengers. Circ Res 1993;73:424–38. Marquez J. Thymosin h10 mRNA expression during gene 1998;17:1395–413. 40. Bustelo XR. Regulatory and signaling properties of early postimplantation mouse development. FEBS Lett 37. Spaargaren M, Bischoff JR, McCormick F. Signal the Vav family. Mol Cell Biol 2000;20:1461–77. 1996;394:103–6. transduction by Ras-like GTPases: a potential target for 41. Huff T, Muller CS, Otto AM, Netzker R, Han- 44. Leonard DM. Ras farnesyltransferase: a new thera- anticancer drugs. Gene Expr 1995;4:345–56. nappel E. h-Thymosins, small acidic peptides with peutic target. J Med Chem 1997;40:2971–90.

www.aacrjournals.org 147 Cancer Res 2005; 65: (1). January 1, 2005

Seung-Hoon Lee, Myung Jin Son, Sun-Hee Oh, et al.

Cancer Res 2005;65:137-148.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/65/1/137

Cited articles This article cites 44 articles, 13 of which you can access for free at: http://cancerres.aacrjournals.org/content/65/1/137.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/65/1/137. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.