Sustained expression of early growth response protein-1 blocks angiogenesis and tumor growth

Abstract

Transient induction of the transcription factor early growth response protein-1 (EGR-1) plays a pivotal role in the transcriptional response of endothelial cells to the angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which are produced by most tumors and are involved in the angiogenic switch. We report here that sustained expression of EGR-1 by recombinant adenoviruses in endothelial cells, however, leads to the specific induction of potent feedback inhibitory mechanisms, including strong up-regulation of transcriptional repressors, negative cell cycle check point effectors, proteins with established antiangiogenic activity, and several proapoptotic genes. Sustained EGR-1 expression consistently leads to an antiangiogenic state characterized by an altered responsiveness to VEGF and bFGF and a striking inhibition of sprouting and tubule formation in vitro. Furthermore, EGR-1-expressing viruses potently inhibit cell invasion and vessel formation in the murine Matrigel model and repress tumor growth in a murine fibrosarcoma model. We propose that gene therapy involving sustained EGR-1 expression may constitute a novel therapeutic principle in the treatment of cancer due to the simultaneous induction of multiple pathways of antiangiogenesis, growth arrest, and apoptosis induction in proliferating cells leading to preferential inhibition of angiogenesis and tumor growth.

Figure 1

Effects of sustained EGR-1 expression in endothelial cells. HUVEC were infected with EGR-1 adenoviruses or control viruses for 24 hours or as indicated, and mRNA was isolated from uninduced samples or cultures induced with VEGF and bFGF for 1 to 3 hours and analyzed using real-time RT-PCR. EGR-1 and VEGFR-1 protein levels were determined by Western blotting in (B) and (D), respectively. A, EGR-1 mRNA and (B) EGR-1 protein produced from the EGR-1 adenovirus. C, up-regulation of VEGFR-1 mRNA by EGR-1 virus. D, up-regulation of VEGFR-1 protein by EGR-1 and inhibition by Ad.NAB2. ns, unspecific band. E, loss of VEGF/bFGF inducibility of uPA mRNA through EGR-1 expression.

Figure 2

Inhibition of tubule and sprout formation in angiogenesis models in vitro and induction of apoptosis. A, in vitro Matrigel model: HUVEC were infected with EGR-1 and control GFP adenoviruses and seeded in parallel on either Matrigel- or gelatin-coated tissue culture plates 24 hours after infection. Sixteen hours thereafter, cultures were assessed by phase-contrast microscopy (top, Matrigel-coated plates; bottom, gelatin-coated plates). Right, the total length of tubules formed was measured. B, in vitro sprout formation assay in fibrin gels: HUMEC infected for 24 hours were seeded onto Cytodex beads, incubated overnight, and then incorporated into fibrin gels in the presence or absence of VEGF or a mixture of VEGF and bFGF. Left, after 48 hours, the cytoskeleton and nuclei were stained with rhodamine-phalloidin (red) and Hoechst dye 333258 (blue), respectively, and evaluated by phase-contrast microscopy; right, the numbers of sprouts were scored to quantitate percentage inhibition. Three independent experiments with duplicate samples were done for both assays. Columns, mean; bars, SD. C, apoptosis induction in EGR-1 virus-infected cells 72 hours after infection: HUVEC seeded on gelatin-coated plates were infected with the viruses. Left, 48 hours after infection, cells started to show increasing signs of apoptosis, such as formation of apoptotic bodies, which led to a reduction in cell number of >50% at 72 hours. Inset, arrow, the formation of apoptotic bodies was displayed by staining with Hoechst dye. Right, apoptotic cell death was confirmed by DNA laddering assays 72 hours after infection. Survival was partially restored by coinfection with Ad.NAB2.

Figure 3

Inhibition of angiogenesis and tumor growth in vivo. A, murine Matrigel model: Ad.EGR-1 and Ad.GFP (1.5 × 10⁸ pfu/mL) were added to Matrigel solution with or without addition of VEGF (300 ng/mL) and injected s.c. into mice. After 6 days, sections were prepared from the plugs and stained with H&E (HE; top) or with anti-CD31 antibodies and DAPI (bottom). Left to right, the images display first the rim of cells regularly formed around the Matrigel plug followed by a section through the plug. Arrows, the border between the rim and the Matrigel. B, total cells in sections of the Matrigel plug were quantified as described in Materials and Methods. Columns, mean of two experiments in duplicates; bars, SD. C, murine fibrosarcoma model: MethA fibrosarcoma cells were injected s.c. into the back of C57Bl/6J mice. On day 4 and, again, on day 6, fibrosarcomas were inoculated with EGR-1 adenoviruses or control GFP viruses. On day 9, tumors were resected, tumor masses were determined, and sections were prepared for histochemical analysis. Cryosections were either stained with hematoxylin (top), rat anti-mouse VEGFR-2 (middle), or rabbit anti-human EGR-1 antibodies and DAPI (bottom). D, reductions in fibrosarcoma tumor weight (top) and VEGFR-2 staining (bottom). Columns, mean of three experiments with three animals per group; bars, SD. Flourescence intensities of VEGR-2 staining of individual sections of the tumors were measured as described in Materials and Methods.