Overexpression and role of the ATPase and putative DNA helicase RuvB-like 2 in human hepatocellular carcinoma.

UNLABELLED: Using a proteomic analysis of human hepatocellular carcinoma (HCC), we identified the overexpression in 4 tumors of RuvB-like 2 (RUVBL2), an ATPase and putative DNA helicase known to interact with beta-catenin and cellular v-myc myelocytomatosis viral oncogene homolog (c-myc). RUVBL2 expression was further analyzed in tumors with quantitative reverse-transcription polymerase chain reaction analysis and immunohistochemistry; in addition, RUVBL2 expression in a HuH7 cell line was silenced by small interfering RNA or increased with a lentiviral vector. RUVBL2 messenger RNA overexpression was confirmed in 72 of 96 HCC cases, and it was associated with poorly differentiated tumors (P = 0.02) and a poor prognosis (P = 0.02) but not with beta-catenin mutations or c-myc levels. Although RUVBL2 was strictly nuclear in normal hepatocytes, tumoral hepatocytes exhibited additional cytoplasmic staining. There was no mutation in the coding sequence of RUVBL2 in 10 sequenced cases. Silencing RUVBL2 in HuH7 HCC cells reduced cell growth (P < 0.001) and increased apoptosis, as shown by DNA fragmentation (P < 0.001) and caspase 3 activity (P < 0.005). This was associated with an increased expression of several proapoptotic genes and with an increased conformational activation of Bak-1 and Bax. On the other hand, HuH7 cells with an overexpression of RUVBL2 grew better in soft agar (P < 0.03), had increased resistance to C2 ceramide-induced apoptosis (P < 0.001), and gave rise to significantly larger tumors when injected into immunodeficient Rag2/gammac mice (P = 0.016). CONCLUSION: RUVBL2 is overexpressed in a large majority of HCCs. RUVBL2 overexpression enhances tumorigenicity, and RUVBL2 is required for tumor cell viability. These results argue for a major role of RUVBL2 in liver carcinogenesis.

Dominant-negative fibroblast growth factor receptor expression enhances antitumoral potency of oncolytic herpes simplex virus in neural tumors.

PURPOSE: Oncolytic herpes simplex viruses (HSV) appear to be a promising platform for cancer therapy. However, efficacy as single agents has thus far been unsatisfactory. Fibroblast growth factor (FGF) signaling is important for the growth and migration of endothelial and tumor cells. Here, we examine the strategy of arming oncolytic HSV with a dominant-negative FGF receptor (dnFGFR) that targets the FGF signaling pathway. EXPERIMENTAL DESIGN: A mouse Nf1:p53 malignant peripheral nerve sheath tumor (MPNST) cell line expressing dnFGFR was generated by transfection. The effects of dnFGFR expression on cell growth and migration in vitro and tumor formation in vivo were determined. The dnFGFR transgene was then inserted into oncolytic HSV G47Delta using a bacterial artificial chromosome construction system. Antitumoral and antiangiogenic activities of bG47Delta-dnFGFR were examined. RESULTS: MPNST 61E4 cells expressing dnFGFR grew less well than parental control cells. bG47Delta-dnFGFR showed enhanced killing of both tumor (human U87 glioma and F5 malignant meningioma cells and murine MPNST 61E4 and 37-3-18-4 cells) and proliferating endothelial cells (human umbilical vascular endothelial cell and Py-4-1) in vitro compared with the control vector bG47Delta-empty without inhibiting viral replication. In vivo, bG47Delta-dnFGFR was more efficacious than its nonexpressing parent bG47Delta-empty at inhibiting tumor growth and angiogenesis in both human U87 glioma and mouse 37-3-18-4 MPNST tumors in nude mice. CONCLUSIONS: By using multiple therapeutic mechanisms, including destruction of both tumor cells and tumor endothelial cells, an oncolytic HSV encoding dnFGFR enhances antitumor efficacy. This strategy can be applied to other oncolytic viruses and for clinical translation.

Prox1 promotes lineage-specific expression of fibroblast growth factor (FGF) receptor-3 in lymphatic endothelium: a role for FGF signaling in lymphangiogenesis.

Fibroblast growth factors play important roles in angiogenesis, but their functions in lymphangiogenesis remain poorly understood. The homeodomain transcription factor Prox1 is essential for development of the lymphatic system by specifying lymphatic endothelial cell (LEC) fate. Here, we identify fibroblast growth factor (FGF) receptor (FGFR)-3 as a novel Prox1 target gene. Ectopic overexpression of Prox1 in blood vascular endothelial cells up-regulates FGFR-3. Prox1 induces the expression of the IIIc isoform, which we also found to be the major isoform of FGFR-3 expressed in LECs. This transcriptional activation is mediated by a direct binding of Prox1 to newly identified Prox1-response elements in the FGFR-3 promoter. Consistently, FGFR-3 is up-regulated in Prox1-positive newly formed lymphatic vessels during embryogenesis and its lymphatic-specific expression is maintained throughout development. We also found that FGF-1 and FGF-2 promote proliferation, migration, and survival of cultured LECs without involvement of vascular endothelial cell growth factor receptor-3. We show that FGF-2 binds to low- and high-affinity receptors on LECs and is efficiently internalized and processed. Moreover, functional inhibition of FGFR-3 using small interfering RNA represses LEC proliferation. Together, these results indicate that FGFR-3 is an initial target of Prox1 during the lymphatic cell fate specification and that FGF signaling may play an important role in lymphatic vessel development.

Design, synthesis, and evaluation of original carriers for targeting vascular endothelial growth factor receptor interactions.

PURPOSE: Angiogenesis is a key event in tumor growth and metastasis, chronic inflammatory disease, and cardiovascular disease. It is controlled by positive and negative regulators, which include vascular endothelial growth factor (VEGF) as the most active of these. VEGF/VEGF receptors are important targets not only for therapy but also for imaging. Based on the structural study of VEGF, we developed a novel cyclopeptide (cyclo-VEGI) that exhibits powerful antitumor properties. We herein report the design of novel molecules derived from cyclo-VEGI as potential targeting agents in cancer and other angiogenesis-related diseases. METHODS: We performed selective chemical modification of the most active VEGF-derived cyclopeptide (cyclo-VEGI). Original hydrophilic linkers were synthesized and coupled to cyclo-VEGI. These reactions provide nanocarriers for delivery. The inhibitory effect of the different compounds on VEGF binding was evaluated in competition assays with 125I-VEGF. A fluorescent cyclo-VEGI peptide was synthezised to assess direct binding and internalization of cyclo-VEGI. RESULTS: Chemical modifications of cyclo-VEGI do not diminish the biological activity of cyclo-VEGI as measured in competition assays; in fact, it is even increased. Moreover there is a strong cellular accumulation of the fluorescent-labeled cyclo-VEGI. Conjugates synthesized in this study may be useful leads to design delivery systems for targeting approaches in cancer and other angiogenesis-related diseases. CONCLUSION: The modified cyclo-VEGIs may have a wide range of applications and represent a useful tool to develop delivery/carrier systems for therapeutic targeting or imaging.

Combinatorial administration of molecules that simultaneously inhibit angiogenesis and invasion leads to increased therapeutic efficacy in mouse models of malignant glioma.

PURPOSE: We investigated the ability of the combinatorial administration of different inhibitors with activities on glioma angiogenesis, migration, and proliferation to produce a prolonged inhibition of glioma growth. EXPERIMENTAL DESIGN: We combined inhibitors affecting solely tumor angiogenesis (PF-4/CTF, cyclo-VEGI) or inhibitors affecting both angiogenesis and invasion together (PEX, PF-4/DLR). RESULTS: When administered in combination, these drugs produced a prolonged and increased inhibition of glioma growth independently from the type of inhibitor used. The combinatory administration was more effective than the administration of a single inhibitor alone, and a strong therapeutic response was reached with a significantly lower amount of protein. The strongest inhibition was observed when human PEX and PF-4/DLR, which affect both glioma angiogenesis and invasion by separate mechanisms, were combined. CONCLUSIONS: This supports the concept that prolonged glioma growth inhibition can be achieved by simultaneous delivery of molecules that target both tumor and endothelial cells and acting by separate mechanisms.

Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells.

Endothelial progenitor cells (EPCs) were shown to be present in systemic circulation and cord blood. We investigated whether EPCs display specific properties compared with mature endothelial cells. Human cord blood CD34+ cells were isolated and adherent cells were amplified under endothelial conditions. Expression of specific markers identified them as endothelial cells, also called endothelial progenitor-derived cells (EPDCs). When compared to mature endothelial cells, human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (HBMECs), endothelial markers, were expressed to the same extent except for KDR, which is expressed more in EPDCs. They display a higher proliferation potential. Functional studies demonstrated that EPDCs were more sensitive to angiogenic factors, which afford these cells greater protection against cell death compared with HUVECs. Moreover, EPDCs exhibit more hematopoietic supportive activity than HUVECs. Finally, studies in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice demonstrated that human circulating EPCs are able to colonize a Matrigel plug. EPDCs display the morphology and phenotype of endothelial cells. Their functional features indicate, however, that although these cells have undergone some differentiation steps, they still have the properties of immature cells, suggesting greater tissue repair capabilities. Future use of in vitro amplified peripheral blood EPDCs may constitute a challenging strategy for cell therapy.

Structure and inhibitory effects on angiogenesis and tumor development of a new vascular endothelial growth inhibitor.

Blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. We describe here the structure and the biological action of a new cyclic peptide derived from vascular endothelial growth factor (VEGF). This 17-amino acid molecule designated cyclopeptidic vascular endothelial growth inhibitor (cyclo-VEGI, CBO-P11) encompasses residues 79-93 of VEGF which are involved in the interaction with VEGF receptor-2. In aqueous solution, cyclo-VEGI presents a propensity to adopt a helix conformation that was largely unexpected because only beta-sheet structures or random coil conformations have been observed for macrocyclic peptides. Cyclo-VEGI inhibits binding of iodinated VEGF165 to endothelial cells, endothelial cells proliferation, migration, and signaling induced by VEGF165. This peptide also exhibits anti-angiogenic activity in vivo on the differentiated chicken chorioallantoic membrane. Furthermore, cyclo-VEGI significantly blocks the growth of established intracranial glioma in nude and syngeneic mice and improves survival without side effects. Taken together, these results suggest that cyclo-VEGI is an attractive candidate for the development of novel angiogenesis inhibitor molecules useful for the treatment of cancer and other angiogenesis-related diseases.

Domain swapping in a COOH-terminal fragment of platelet factor 4 generates potent angiogenesis inhibitors.

A few peptide residues in structurally important locations often determine biological functions of proteins implicated in the regulation of angiogenesis. We have shown recently that the short COOH-terminal segment PF-4(47-70) derived from platelet factor 4 (PF-4) is the smallest sequence that conserves potent antiangiogenic activity in vitro and in vivo. Here we show that modified COOH-terminal PF-4 peptides containing the sequence ELR (or related DLR), a critical domain present in proangiogenic chemokines, surprisingly elicit several times greater antiangiogenic potential than the original peptide. The modified peptides inhibit binding of iodinated vascular endothelial growth factor and fibroblast growth factor 2 to endothelial cell receptors, endothelial cell proliferation, migration, and microvessel assembly in the rat aortic ring model at lower doses than PF-4(47-70). On the differentiated chick chorioallantoic membrane, topical application of 40 micro g of modified peptides potently reduces capillary angiogenesis induced by vascular endothelial growth factor(165), a dose where peptide PF-4(47-70) was inactive. Established intracranial glioma in nude mice decreased significantly in size when treated locally with a total dose of 250 micro g of peptide PF-4(47-70)DLR (n = 10) compared with the same dose of the original PF-4(47-70) peptide (n = 10) or controls (n = 30). Tailored PF-4 peptides represent a new class of antiangiogenic agents with a defined mode of action and a strong in vivo activity.