[CANCER RESEARCH 63, 8968–8976, December 15, 2003]Repression of Vascular Endothelial Growth Factor A in Glioblastoma Cells UsingEngineered Zinc Finger Transcription FactorsAndrew W. Snowden, Lei Zhang, Fyodor Urnov, Carolyn Dent, Yann Jouvenot, Xiaohong Zhong, Edward J. Rebar,Andrew C. Jamieson, H. Steven Zhang, Siyuan Tan, Casey C. Case, Carl O. Pabo, Alan P. Wolffe, andPhilip D. GregorySangamo BioSciences, Inc., Richmond, CaliforniaABSTRACTAngiogenic factors are necessary for tumor proliferation and thus areattractive therapeutic targets. In this study, we have used engineered zincfinger protein (ZFP) transcription factors (TFs) to repress expression ofvascular endothelial growth factor (VEGF)-A in human cancer cell lines.We create potent transcriptional repressors by fusing a designed ZFPtargeted to the VEGF-A promoter with either the ligand-binding domainof thyroid hormone receptor␣or its viral relative, vErbA. Moreover, thisZFP-vErbA repressor binds its intended target site in vivo and mediatesthe specific deacetylation of histones H3 and H4 at the targeted promoter,a result that emulates the natural repression mechanism of these domains.The potential therapeutic relevance of ZFP-mediated VEGF-A repressionwas addressed using the highly tumorigenic glioblastoma cell line U87MG.Despite the aberrant overexpression of VEGF-A in this cell line, engi-neered ZFP TFs were able to repress the expression of VEGF-A by>20-fold. The VEGF-A levels observed after ZFP TF-mediated repressionwere comparable to those of a nonangiogenic cancer line (U251MG),suggesting that the degree of repression obtained with the ZFP TF wouldbe sufficient to suppress tumor angiogenesis. Thus, engineered ZFP TFsare shown to be potent regulators of gene expression with therapeuticpromise in the treatment of disease.INTRODUCTIONAngiogenesis, the process by which new capillary blood vesselsgrow, is of critical importance to tumor progression and therefore is anattractive target in the treatment of cancer (1). Solid tumors, forexample, require neovascularization for growth beyond a diameter of⬃3 mm (2), and this angiogenic response is correlated with increasedtumorigenicity (3). Vascular endothelial growth factor (VEGF)-A,which is a potent effector of angiogenesis, is overexpressed in avariety of human cancers, including highly invasive brain tumors (4).Expression of VEGF-A is usually tightly regulated in response tophysiological conditions. Up-regulation (which occurs in response tohypoxia) can induce the proliferation of capillary endothelial cells,leading to remodeling of pre-existing capillaries or the growth ofadditional capillary networks (5). Constitutive high-level VEGF-Aexpression, as seen in many human cancers, provokes aberrant re-modeling and capillary growth and thus facilitates tumor progression.Additionally, VEGF-mediated capillary and endothelial remodelingappear to be integral steps in facilitating tumor metastasis via the hostblood supply (6, 7). Conversely, the inhibition of VEGF-inducedangiogenesis has been shown to suppress tumor growth in vivo (8).Repression or inhibition of VEGF-A function thus presents a potentialavenue for anticancer therapy.Misregulation of gene expression leading to constitutively highVEGF-A levels, for example, is a hallmark of cancer (1). Tumor-specific transcriptional activity has been linked to the aberrant actionof chromatin-based modulators of gene regulation typified by histonedeacetylation activities in leukemia (9) and histone methyltransferaseenzymes in prostate cancer (10). Indeed, a cancer-specific transcrip-tional circuit is evidenced in the up-regulation of VEGF-A in glio-blastoma (11). Treatments that correct such defects at a transcriptionallevel are limited. Engineered zinc finger protein (ZFP) transcriptionfactors (TFs) represent a novel class of potential therapeutic agentsthat invoke the natural mechanisms of gene control and offer theability to regulate a specific gene at its endogenous location in thegenome (12). In this regard, we have previously used engineered ZFPTFs to specifically up-regulate the expression of the endogenousVEGF-A gene both in human cell lines (13) and in whole animalmodels of angiogenesis (14). It remained unclear, however, whether adesigned ZFP TF repressor would be sufficient to reduce the highlevels of VEGF expression associated with vascularizing tumors to atherapeutically relevant degree. In the present study, we have nowused these DNA-binding proteins to inhibit VEGF-A expression in avariety of human cancer cell lines. We show that ZFP TFs can repressVEGF-A expression at the protein and mRNA levels in HEK293 cells.To address the therapeutic potential of ZFP TF-mediated VEGF-Arepression, we investigated the potency of this reagent in the highlytumorigenic human glioblastoma line U87MG. Using a stableU87MG cell line engineered to exhibit inducible expression of theZFPA-vErbA protein, we demonstrate that expression of VEGF-A canbe repressed to a level that parallels that of a nonangiogenic, low-tumorigenic glioblastoma line (U251MG). These data establish thatspecific engineered ZFP TFs can potently inhibit the expression ofVEGF-A in a therapeutically relevant model system.MATERIALS AND METHODSCell Culture and Transient Transfections. HEK293 cells were grown inDMEM supplemented with 10% fetal bovine serum in a 5% CO2incubator at37°C. Transfection experiments were carried out as described previously (15).Charcoal-filtered fetal bovine serum (HyClone Laboratories) was used asindicated. HEK293 cells were adapted to growth in charcoal-filtered serum for1 week before use. Cell proliferation and viability were determined by theWST-1 assay (Roche), according to the manufacturer’s recommendations.Secreted VEGF-A Protein ELISA. Secreted VEGF-A in the tissue culturemedia by transfected HEK293 cells was assayed after 48 h using a humanVEGF-A ELISA kit (R&D Systems) in duplicate according to the manufac-turer’s recommendations.Assembly of Expression Constructs Carrying ZFP-TR␣and ZFP-v-ErbA Chimeras. The ligand-binding domain of chicken (Gallus gallus) thy-roid hormone receptor␣1 (TR␣1; NR1A1) was amplified from a clone offull-length TR␣1 in pT7TS (16) using the following primers: forward primer,5⬘-AAG-GAT-CCA-TCT-CCG-TGG-GCA-TGG-CCA-TGG-3⬘; and reverseprimer, 5⬘-AAA-AGC-TTA-CAC-CTC-CTG-GTC-CTC-GAA-GACC-3⬘.These primers isolate amino acids 114–408 of the receptor (GenBank acces-sion number M24748),