Hui Guan

A small interfering RNA targeting vascular endothelial growth factor inhibits Ewing's sarcoma growth in a xenograft mouse model.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewings sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewings sarcoma.

Osterix, a transcription factor for osteoblast differentiation, mediates antitumor activity in murine osteosarcoma

Osterix is a novel zinc finger-containing transcription factor that is essential for osteoblast differentiation and bone formation. We hypothesized that osterix might have a role in osteosarcoma tumor growth and metastasis. Northern blot analysis showed that the mRNA level of osterix was decreased in two mouse osteosarcoma cell lines compared with its level in normal mouse osteoblasts. Osterix expression was also decreased in three human osteosarcoma cell lines. Transfection of the osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Osterix expression was also associated with decreased osteolysis. Using an in vitro migration assay, osterix suppressed the migration of tumor cells to lung extracts. These results suggest that osterix expression may play a role in osteosarcoma tumor growth and metastasis.

Fas-negative osteosarcoma tumor cells are selected during metastasis to the lungs: the role of the Fas pathway in the metastatic process of osteosarcoma.

Low expression of Fas by different tumors including osteosarcoma, correlates with poor prognosis. We found that osteosarcoma lung metastases from patients expressed negligible amounts of Fas, but primary tumors often expressed high Fas levels. The reason for this discrepancy is unknown. We hypothesized that because FasL is constitutively expressed in the lungs, Fas-positive (Fas+) tumor cells entering the lungs would bind with FasL and die from Fas-induced apoptosis, resulting in the “selection” of Fas-negative (Fas−) cells, which would eventually form metastases. To test this hypothesis, we injected K7 osteosarcoma cells, which express functional Fas in vitro, into mice and confirmed that its bone tumors were Fas+, but lung metastases were Fas−. Next, to inhibit Fas signaling without affecting Fas expression, we transfected these cells with a FADD-dominant negative (FDN) plasmid and developed K7/FDN cells. Metastases formed by K7/FDN cells contained Fas+ tumor cells. Moreover, K7/FDN cells were retained in the lungs longer and formed more lung metastases than K7 cells. In addition, the incidence of lung metastases in FasL-deficient mice injected with K7 cells was higher than that in wild-type mice. Metastases from FasL-deficient mice but not from wild-type mice contained Fas+ tumor cells. Based on that, we conclude that Fas− osteosarcoma cells are selected during lung metastases formation and that inhibition of Fas signaling in tumors or lack of FasL in the host environment allows the proliferation of Fas+ osteosarcoma cells in the lungs and promotes metastases growth. Therefore, Fas may be considered as a new therapeutic target for osteosarcoma treatment. (Mol Cancer Res 2007;5(10):991–9)

Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into ewing sarcoma tumors

This study evaluated the therapeutic efficacy of interleukin 12 (IL‐12) gene therapy in Ewing sarcoma and whether murine mesenchymal stem cells (MSCs) could serve as vehicles for IL‐12 gene delivery.

Production of VEGF165 by Ewing's sarcoma cells induces vasculogenesis and the incorporation of CD34+ stem cells into the expanding tumor vasculature

The Ewings sarcoma cell line TC71 overexpresses vascular endothelial growth factor isoform 165 (VEGF165), a potent proangiogenic molecule that induces endothelial cell proliferation, migration, and chemotaxis. CD34+ bone marrow stem cells can differentiate into endothelial and hematopoietic cells. We used a transplant model to determine whether CD34+ cells migrate from the bone marrow to Ewings sarcoma tumors and participate in the neovascularization process that supports tumor growth. We also examined the role of VEGF165 in CD34+ cell migration. Human umbilical cord CD34+ cells were transplanted into sublethally irradiated severe combined immunodeficient mice. Seven days later, the mice were injected subcutaneously with TC71 tumor cells. Tumors were excised 2 weeks later and analyzed by immunohistochemistry. The tumor sections expressed both human VE‐cadherin and mouse CD31, indicating involvement of donor‐derived human cells in the tumor vessels. To determine the role of VEGF165 in the chemoattraction of CD34+ cells, we generated two VEGF165‐deficient TC71 clones, a stable anti‐sense VEGF165 cell line (Clone 17) and a VEGF165 siRNA‐inhibited clone (TC/siVEGF7‐1). The resulting VEGF165‐deficient tumor cells had normal growth rates in vitro, but had delayed growth when implanted into mice. Immunohistochemical analysis revealed decreased infiltration of CD34+ cells into both VEGF165‐deficient tumors. These data show that bone marrow stem cells contribute to the growing tumor vasculature in Ewings sarcoma and that VEGF165 is critical for the migration of CD34+ cells from the bone marrow into the tumor.

Herceptin Down-Regulates HER-2/neu and Vascular Endothelial Growth Factor Expression and Enhances Taxol-Induced Cytotoxicity of Human Ewing's Sarcoma Cells In vitro and In vivo

We have previously shown that high levels of HER-2/neu protein were overexpressed in human Ewings sarcoma cells (TC71, SK-ES1) relative to normal human osteoblasts. The purpose of this study was to determine whether herceptin alone or in combination with chemotherapeutic agents could inhibit the growth of Ewings sarcoma in vitro and in vivo. Western blot analysis showed that the protein levels of HER-2/neu were decreased following herceptin treatment. Cell growth was also inhibited by herceptin in a dose-dependent manner with an IC50 of 4 mg/mL in TC71 and SK-ES1 cell line, whereas human immunoglobin had no effect. Northern blot and ELISA showed the RNA expression and protein levels of vascular endothelial growth factor were also inhibited by herceptin treatment with no alteration in HIF-1α protein and topoisomerase IIα expression. Furthermore, Ewings sarcoma tumor growth was significantly delayed by 100 mg/kg herceptin treatment in our Ewings sarcoma xenograft mouse model. Combining taxol with herceptin resulted in additive cytotoxicity, whereas herceptin-etoposide, doxorubicin, and 9-nitrocamptothecin combinations did not. Taxol-herceptin enhanced growth inhibition in TC71 cells in vitro compared with either agent alone. Ewings sarcoma growth was also delayed in vivo and mean tumor size was significantly lower in mice treated with herceptin plus taxol than in those receiving taxol or herceptin alone. These data suggest that herceptin in combination with taxol may be a therapeutic option in the treatment of Ewings sarcoma.

Delta-Like Ligand 4 Plays a Critical Role in Pericyte/Vascular Smooth Muscle Cell Formation during Vasculogenesis and Tumor Vessel Expansion in Ewing's Sarcoma

Purpose: Bone marrow (BM) cells contribute to tumor vessel formation that supports the growth of Ewings sarcoma. These BM cells migrate into the tumor and differentiate into endothelial cells and pericytes. We investigated whether delta-like ligand 4 (DLL4) played a role in the formation of BM-derived pericytes/vascular smooth muscle cells (vSMC) during tumor vessel formation. Experimental Design: Using immunohistochemistry, we examined the expression pattern of DLL4 in 14 patient samples and two xenograft mouse models of Ewings sarcoma. We then used intratumor injections of short hairpin RNA to inhibit DLL4 expression in Ewings sarcoma tumors in mice, and evaluated the effect on BM-derived pericytes/vSMCs. Results: DLL4 was expressed by perivascular cells in 12 of 14 human samples and in BM-derived pericytes/vSMCs in both A4573 and TC71 xenograft tumors. Inhibition of DLL4 expression by short hairpin RNA correlated with the decreased numbers of BM-derived cells in tumor vessels and the decreased numbers of α-SMA+, desmin+, and NG2+ pericytes/vSMCs, as well as increased tumor hypoxia. Conclusions: DLL4 is important for the formation of BM-derived pericytes/vSMCs during vasculogenesis in Ewings sarcoma. DLL4 may be a therapeutic target for treatment of Ewings sarcoma by inhibition of blood vessel formation. Clin Cancer Res; 16(3); 848–56

Suppression of Ewing's Sarcoma Tumor Growth, Tumor Vessel Formation, and Vasculogenesis Following Anti–Vascular Endothelial Growth Factor Receptor-2 Therapy

Purpose: We previously showed that bone marrow cells participate in new tumor vessel formation in Ewings sarcoma, and that vascular endothelial growth factor 165 (VEGF165) is critical to this process. The purpose of this study was to determine whether blocking VEGF receptor 2 (VEGFR-2) with DC101 antibody suppresses tumor growth, reduces tumor vessel formation, and inhibits the migration of bone marrow cells into the tumor. Experimental Design: An H-2 MHC-mismatched bone marrow transplant Ewings sarcoma mouse model was used. Bone marrow cells from CB6F1 (MHC H-2b/d) mice were injected into irradiated BALB/cAnN mice (MHC H-2d). TC71 Ewings sarcoma cells were s.c. injected 4 weeks after the bone marrow transplantation. Mice were then treated i.p. with DC101 antibody or immunoglobulin G (control) twice a week for 3 weeks starting 3 days after tumor cell injection. Results: DC101 antibody therapy significantly reduced tumor growth and tumor mean vessel density (P < 0.05) and increased tumor cell apoptosis. Decreased bone marrow cell migration into the tumor was also shown after DC101 therapy as assessed by the colocalization of H-2Kb and CD31 using immunohistochemistry. DC101 inhibited the migration of both human and mouse vessel endothelial cells in vitro. Conclusion: These results indicated that blocking VEGFR-2 with DC101 antibodies may be a useful therapeutic approach for treating patients with Ewings sarcoma.

VEGF165 promotes the osteolytic bone destruction of Ewing's sarcoma tumors by upregulating RANKL

The purpose of this study was to determine whether vascular endothelial growth factor-165 (VEGF165) contributed to the osteolytic process in Ewings sarcoma. VEGF165 induced osteoclast formation from murine bone marrow cells. Tartrate-resistant acid phosphatase (TRAP) staining demonstrated significantly fewer osteoclasts in VEGF-inhibited TC/siVEGF7-1 tumors compared to TC71 parental or TC/si-control tumors. Receptor activator NF-kappaB (RANKL), a critical osteoclastogenic factor, was decreased in TC/siVEGF7-1 cells. Incubation of these cells with recombinant VEGF165 upregulated RANKL in a dose- and time-dependent manner. The induction of (RANKL) by VEGF165 was also demonstrated in MC3T3-E1 mouse osteoblast cells and bone marrow stromal cells. This upregulation was transcriptionally mediated by an effect on the RANKL promoter. Both VEGF and EWS/FLI-1 increased RANKL promoter activity. Taken together, these data suggest that modulation of RANKL by VEGF165 may be one of the mechanisms responsible for the osteolytic process induced by Ewings sarcoma cells. VEGF165 may, therefore, play an important role in modulating RANKL gene expression in the bone marrow microenvironment during the metastatic process, thereby contribution to tumor induced bone lysis.

VEGF165, but not VEGF189, stimulates vasculogenesis and bone marrow cell migration into Ewing's sarcoma tumors in vivo

We previously showed that bone marrow stem cells participate in the tumor vessel expansion that supports the growth of Ewings sarcoma tumors in vivo. The purpose of this study was to determine the relative importance of two isoforms of vascular endothelial growth factor (VEGF) in tumor vessel expansion and recruitment of bone marrow–derived cells during tumor growth. We injected VEGF165-siRNA–transfected cells (TCsi/7-1), control siRNA–transfected cells (TC/si-control), or TC71 parental Ewings sarcoma cells into nude mice. The TCsi/7-1 tumors were then treated with adenoviral vectors expressing VEGF165 (Ad-VEGF165), VEGF189 (Ad-VEGF189), or β-galactosidase (Ad-β-gal). Bone marrow cells labeled with fluorescent tracker dye were injected into the mice 3 weeks later. The TCsi/7-1 tumors were significantly smaller (P < 0.05), had decreased vessel density, and showed significantly lower bone marrow cell migration than did TC71 parental and TC/si-control tumors. Treatment with Ad-VEGF165, but not Ad-VEGF189 or Ad-β-gal, resulted in a significant increase in bone marrow cell infiltration, tumor vessel density, and tumor growth. Immunohistochemical staining revealed that the injected bone marrow cells migrated to and incorporated into the expanding CD31+ tumor vessel network. Taken together, these data show that VEGF165 is a chemoattractant that recruits bone marrow cells into the tumor area. These data provide a mechanism by which Ewings sarcoma cells induce vasculogenesis. (Mol Cancer Res 2007;5(11):1125–32)