Repression of Vascular Endothelial Growth Factor Expression by the Zinc Finger Transcription Factor ZNF24

Research Article

Jay Harper,1,2 Li Yan,4 Robyn M. Loureiro,3 Iinmin Wu,1,2 Jianmin Fang,5 Patricia A. D’Amore,3 and Marsha A. Moses1,2

1Vascular Biology Program, Children’s Hospital Boston and 2Department of Surgery and 3Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; 4Oncology Research, Centocor R&D, Inc., Malvern, Pennsylvania; and 5Department of Preclinical Oncology and Immunology, Cell Genesys, Inc., San Francisco, California

Abstract sequestering Sp1 (9) and by binding to HIF-1, targeting it for Vascular endothelial growth factor (VEGF) is a potent sti- ubiquitin-mediated degradation (10). p53 also inhibits Sp1- mulator of angiogenesis. Although many positive regulators mediated VEGF expression by targeting it for ubiquitination and of VEGF have been identified, relatively little is known regard- proteosomal degradation (11). p53, as well as the p53-related g ing the negative regulation of VEGF expression. We identified TAp63 , can also repress activity of the VEGF promoter (12, 13). In a zinc finger transcription factor, ZNF24, that may repress the current study, we show that the zinc finger transcription factor VEGF transcription. An inverse correlation between expres- ZNF24 may represent a novel transcriptional repressor of VEGF sion of VEGF and ZNF24 was observed in a series of inde- expression. pendent studies. ZNF24 was up-regulated in angiogenic tumor ZNF24, also known as ZNF191 (14, 15) and KOX17 (16), is a relatively uncharacterized transcription factor belonging to the nodules where VEGF expression is significantly decreased compared with preangiogenic nodules. In human breast car- family of SCAN box domain–containing Kru¨ppel-like Cys2-His2 zinc cinoma cells cultured under normoxic conditions, ZNF24 finger transcription factors (14, 16). It contains four zinc finger levels were significantly up-regulated whereas VEGF levels motifs that encode putative DNA binding domains. The ZNF24 gene were low. In contrast, VEGF was significantly increased in is localized to chromosome 18q12.1 (14–16), a region frequently hypoxic cells whereas ZNF24 was down-regulated. The same deleted in colorectal carcinomas, suggesting a possible role in the inverse correlation between ZNF24 and VEGF was also negative regulation of tumor growth (17, 18). ZNF24 was shown to observed in 70% of matched cDNA pairs of normal and malig- possess transrepression activity of the GAL4 promoter in Chinese nant tissues from human colon and breast biopsies. Over- hamster ovary and NIH-3T3 cells (14); however, little else is known expression of ZNF24 resulted in a significant down-regulation regarding its role in gene expression. of VEGF, whereas silencing of ZNF24 with small interfering In the present study, ZNF24 was identified, via microarray analyses, as a gene that was differentially expressed in two RNA led to increased VEGF expression. Cotransfection of in vivo ZNF24 and a VEGF promoter luciferase reporter construct in independent systems: an tumor model that reliably recapitulates the transition to the angiogenic phenotype (19, 20) MDA-MB-231 cells resulted in a significant decrease in VEGF in vitro promoter activity. Taken together, these data suggest that and an system analyzing hypoxia-inducible gene expres- ZNF24 is involved in negative regulation of VEGF and may sion. In both studies, an inverse correlation between ZNF24 represent a novel repressor of VEGF transcription. [Cancer Res and VEGF was observed. ZNF24 mRNA expression was also 2007;67(18):8736–41] inversely correlated with VEGF expression in matched pairs of human colon and breast tumors compared with adjacent normal tissue. The functional relationship between ZNF24 and VEGF Introduction expression was investigated via ZNF24 overexpression, silencing, Vascular endothelial growth factor (VEGF) is required for and VEGF promoter analyses. Our results suggest that ZNF24 physiologic angiogenesis during development as shown by the may be involved in the negative regulation of VEGF expression and homozygous or heterozygous ablation of the VEGF gene and the that it may function as a novel transcriptional repressor of the results from the use of VEGF antagonists in developmental studies VEGF gene. (1, 2). Increased VEGF expression is associated with many angiogenesis-dependent pathologies, such as cancer (reviewed in ref. 3) and many ocular diseases (4, 5). To date, much research has Materials and Methods been focused on the positive regulation of VEGF expression. Several Tumor model and cell culture. Preangiogenic and angiogenic tumors transcriptional activators of VEGF are known, including Sp1 (6, 7) were established and harvested as previously described (19, 20). Human and hypoxia-inducible factor-1 (HIF-1; reviewed in ref. 8), yet few MDA-MB-231 and MCF-7 breast carcinoma cells and U87 MG glioblastoma negative regulators of VEGF have been identified. The von Hippel- cells (American Type Culture Collection) were cultured in DMEM Lindau tumor suppressor indirectly inhibits VEGF expression by (Invitrogen/Life Technologies) supplemented with 10% bovine calf serum (HyClone) and 1% glutamine/penicillin/streptomycin (Invitrogen/Life

Technologies) at 37j,5%CO2. For hypoxia experiments, MDA-MB-231 cells were cultured in a hypoxia chamber (Model MIC-101, Billups-Rothenberg,

Requests for reprints: Marsha A. Moses, Department of Surgery, Harvard Medical Inc.) in 1% O2,5%CO2, and 94% N2 for 24 h. For conditioned media School, Vascular Biology Program, Children’s Hospital Boston, 300 Longwood Avenue, experiments, cells were washed with PBS, and the media changed to serum- Boston, MA 02115-5737. Phone: 617-919-2207; Fax: 617-730-0231; E-mail: marsha. free DMEM, 1% glutamine/penicillin/streptomycin, 24 h before collection. [email protected]. I2007 American Association for Cancer Research. Conditioned media and/or RNA was harvested 24 h after culture under doi:10.1158/0008-5472.CAN-07-1617 either normoxic or hypoxic conditions.

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Microarray analyses. RNA was purified from tumor nodules and cells protein concentration (Micro BCA protein assay, Pierce) and VEGF using the RNeasy kit (Qiagen) according to manufacturer’s protocol. cDNA concentrations determined by Quantikine human VEGF immunoassay synthesis, cRNA synthesis and labeling, and hybridization to microarrays (R&D Systems). were done by the Gene Array Technology Center at Brigham and Women’s VEGF promoter reporter assays. MDA-MB-231 cells were plated in Hospital (Boston, MA). The human U95A GeneChip array (Affymetrix) and antibiotic-free media, cultured for 24 h, then transiently cotransfected with the MICROMAX human cDNA array (New England Nuclear) were used in equimolar ratios of either a ZNF24 or LacZ expression vector and with these experiments. Analysis of hybridization data was done with GeneChip either VEGF promoter luciferase constructs or a control vectors using 3.1 expression analysis software (Affymetrix). FuGENE 6 (Roche Diagnostics) according to manufacturer’s protocols. After Semiquantitative reverse transcription–PCR. Reverse transcription 48 h, with a medium change at 24 h, the cells were lysed with Passive lysis À was done using SuperScriptII RNase H (Invitrogen) according to buffer (Promega) according to manufacturer’s protocols. Luciferase activity manufacturer’s protocols. Serial dilutions of cDNA were prepared, normal- in lysates was assessed with the Dual luciferase reporter assay system ized against h-actin, then analyzed via PCRto validate microarray results. (Promega) according to manufacturer’s protocols, and results were The following primers were used: ZNF24 primers (F 5¶-GGTTTGAAAGAAA- normalized to transfection efficiency of a third cotransfected thymidine GAGAAATCC-3¶,R5¶-AAGTTTTTCTGCATTGTGTC-3¶; 374-bp product) kinase–driven renilla luciferase vector. and VEGF primers that were designed to identify all isoforms (F 5¶- ACTTTCTGCTGTCTTGGGTGCATT-3¶,R5¶-TCACCGCCTCGGCTTGT- Results CACA-3¶; 421 bp VEGF , 522 bp VEGF , 625bp VEGF ). In PCRof 121 165 189 Inverse correlation between ZNF24 and VEGF. In the first matched cDNA pairs (see below), a second reverse VEGF primer was used: R with the above forward primer (R5 ¶-TTCTTTGGTCTGCATTCACATTTGTT- study, we used a tumor model that reliably recapitulates the in vivo 3¶; 379 bp). h-Actin primers (F 5¶-AGAAGATCTGGCACCACACCTTCTA- transition to the angiogenic phenotype (19). Transcrip- CAAT-3¶,R5¶-ACATAGCACAGCTTCTCTTTAATGTCAC-3¶; 408 bp) were also tional profiling of preangiogenic and angiogenic tumor nodules used. PCRreactions with Platinum PCRSuperMix (Invitrogen) were tested at was done to identify genes that were differentially expressed 94jC 5 min (‘‘hotstart’’), then 94jC 1 min, 60jC 30 s, and 72jC 30 s for 20 to during the transition to the angiogenic phenotype. Analysis of 30 cycles, followed by extension at 72jC 10 min. PCRproducts were analyzed preangiogenic and angiogenic tumor gene expression identified via gel electrophoresis. ZNF24 as a gene that was up-regulated in angiogenic nodules. Real-time quantitative PCR. Real-time quantitative PCR (qPCR) was This result was confirmed via several rounds of sqRT-PCR done done using the DNA Engine Opticon II real-time PCRdetection system (Bio- on normalized serial dilutions of cDNA synthesized from each set Rad Laboratories/MJ Research). cDNA synthesis from preangiogenic and of nodules (Fig. 1A). Conversely, VEGF expression was signifi- angiogenic tumor nodule RNA was done as described above. qPCR ZNF24 primers are F 5¶-ATGTCTGCACAGTCAGTGGAAGAAGATTCA-3¶,R5¶- cantly up-regulated in avascular nodules compared with vascular A CGGAAAATCTCTGGGTCTGGGAGATGGTTC-3¶. qPCRVEGF primers are nodules (Fig. 1 ), an observation that confirms our previously F5¶-GCAGAAGGAGGAGGGCAGAAT-3¶,R5¶-GCACACAGGATGGCTT- reported results (20). The inverse correlation between expression GAAGA-3¶. qPCRglyceraldehyde-3-phosphate dehydrogenase primers are of ZNF24 and VEGF was also confirmed using real-time qPCR F5¶-CAGCCTCAAGATCATCAGCA-3¶,R5¶-GTCTTCTGGGTGGCAGTGAT- (Fig. 1C). 3¶. qPCRreaction with IQ SYBRGreen Supermix (Bio-RadLaboratories) at Interestingly, the same inverse correlation between ZNF24 and hotstart 95j 2 min, then 94jC10s,55jC10s,72jC 10 s for 45 total cycles VEGF was observed in transcriptional profiling experiments aimed with a plate read after each cycle. Upon completion a melting point at identifying hypoxia-inducible changes in gene expression of determination from 55jCto95jC was conducted, reading for 1 s at every MDA-MB-231 human breast carcinoma cells. ZNF24 was down- 0.5jC change in temperature followed by an extension step at 72jC. PCR of matched cDNA pairs. cDNAs from matched pairs of normal and regulated in cells cultured under hypoxia, where VEGF is up- malignant human colon and breast tissues were purchased from Clonetech regulated, and up-regulated in cells cultured under normoxic B and normalized to ribosome binding protein-9. Semiquantitative reverse conditions, where VEGF is down-regulated (Fig. 1 ). The inverse transcription-PCR(sqRT-PCR)was done on these samples with VEGF and correlation between ZNF24 and VEGF, observed in these two ZNF24 primers described above using the reaction hotstart at 94j 5 min, separate and independent systems, suggested that this transcrip- then 93j 30 s, 54j 10 s, and 72j 14 s for 30 cycles, followed by extension at tion factor may be involved in negative regulation of VEGF 72j 4 min. PCRproducts were analyzed via gel electrophoresis. expression. Establishment of MDA-MB-231 clones that overexpress ZNF24. Full- Expression of ZNF24 and VEGF in matched tumor/normal length human ZNF24 was cloned from MDA-MB-231 cells into the cDNA pairs. ZNF24 is localized to chromosome 18q12.1, a region expression vector pcDNA3.1/V5-His-TOPO using the Topo-TA cloning kit that is mutated in a variety of human cancers, including colorectal (Invitrogen) according to manufacturer’s protocols and verified with carcinomas and invasive breast cancer suggesting a potential sequencing. Expression of ZNF24 is under the control of cytomegalovirus (CMV) promoter. The primers used to generate the full-length ZNF24 cDNA: tumor suppressor role for this transcription factor (17, 18, 21). F5¶- CACCATGTCTGCACAGTCAGTGG-3¶,R5¶- AACTTCCACAACATTCA- Based on this finding, matched cDNA pairs of normal and malig- GAA-3¶. The expression vector was transfected into MDA-MB-231 cells with nant colon and breast tissues from human patients were analyzed Effectene (Qiagen) according to manufacturer’s protocols. Clones over- via RT-PCR for differential expression of ZNF24 and VEGF (Fig. 2A). expressing ZNF24 (MDA-MB-231-ZNF24) were selected with G418 In four of five matched pairs from colorectal carcinoma patients, (500 Ag/mL; Invitrogen) and overexpression of ZNF24 verified by sqRT-PCR. an inverse correlation between VEGF and ZNF24 was observed, Silencing of ZNF24 expression. MCF7 human breast carcinoma cells consistent with the data from our angiogenic switch model and the were transiently transfected with 100 nmol/L of ZNF24 small interfering hypoxia-inducible gene expression study. Additionally, ZNF24 was RNAs or control small interfering RNAs (Dharmacon, Inc.) with Dharma- down-regulated in the majority of colorectal carcinoma samples FECT. RNA was purified from transfected cells after 24 to 48 h in culture, compared with normal cohorts and was down-regulated in all five and ZNF24 and VEGF expression was analyzed by sqRT-PCR as described above. breast carcinomas compared with normal mammary tissues. in vitro Determination of VEGF synthesis. MDA-MB-231 and MDA-MB-231- Consistent with the results obtained from our data with ZNF24 cells were cultured under either normoxic or hypoxic conditions, MDA-MB-231 breast carcinoma cell lines, three of five matched as described above, in the presence of 0.1% bovine serum albumin cDNA pairs displayed an inverse correlation of VEGF expression (Intergen). After 24 h, conditioned media samples were normalized against with respect to ZNF24 levels. www.aacrjournals.org 8737 Cancer Res 2007; 67: (18). September 15, 2007

using human VEGF ELISA kits. VEGF protein levels were significantly reduced in multiple MDA-MB-231 clones compared with parental cells (Fig. 3A) or transfection control cells (data not shown). Comparable inhibition of VEGF secretion was observed whether the cells were cultured under normoxic or hypoxic conditions. Silencing of ZNF24 leads to up-regulation of VEGF. To further verify the inverse correlation observed between expression of ZNF24 and VEGF, ZNF24 expression was knocked down with small interfering RNAs. MCF7 breast carcinoma cells, in which VEGF expression level is less than that of the MDA-MB-231 cells (data not shown), were transfected with commercially available small interfering RNAs, targeting ZNF24 (Dharmacon) or with small interfering RNA controls. Transfection of ZNF24 small interfering RNAs resulted in efficient knockdown of ZNF24 expression (Fig. 3B), which correlated with increased VEGF expression, whereas transfection with control small interfering RNAs had no significant effect on ZNF24 or VEGF expression levels. Decreased VEGF promoter activity in response to overexpression of ZNF24. ZNF24 is a Cys2-His2 transcription factor with four Kru¨ppel-like zinc finger motifs that encode putative DNA binding domains. Therefore, the ability of this transcription factor to repress transcription of VEGF was investigated. VEGF promoter luciferase reporter constructs (Fig. 3C) were constructed and used to analyze the effect of overexpression of ZNF24 on VEGF promoter activity. MDA-MB-231 cells were cotransfected with the VEGF promoter construct or control constructs and either ZNF24 or control LacZ expression plasmids. Cotransfection with VEGF promoter constructs and ZNF24 resulted in a significant decrease in VEGF promoter activity as reported by decreased luciferase activity compared with cells that were transfected with the LacZ vector (Fig. 3D). These data indicate that overexpression of ZNF24 results in down-regulation of VEGF promoter activity and suggests that ZNF24 may negatively regulate expression of VEGF via Figure 1. Expression of ZNF24 is inversely correlated with VEGF expression in vitro and in vivo.Results from microarray analyses were verified by transcriptional repression of the VEGF gene. sqRT-PCR using cDNA dilutions normalized to h-actin levels.sqRT-PCR for expression of ZNF24 and VEGF in RNA purified from preangiogenic and angiogenic tumor nodules (A) and from MDA-MB-231 breast carcinoma cells (B) cultured under either normoxic or hypoxic conditions.In each system, increased expression of ZNF24 was correlated with decreased expression of VEGF.The differential expression of ZNF24 and VEGF in preangiogenic tumor nodules was confirmed by qPCR (C).

Data from these matched cDNA pair analyses are consistent with the results from the microarray analyses. The strong inverse correlation between ZNF24 and VEGF expression observed in vitro and in vivo in these multiple systems and the fact that the chromosomal locus of the ZNF24 gene is mutated in several types of cancer suggest that ZNF24 may represent a potential tumor suppressor that may function to negatively regulate the expression of VEGF. Down-regulation of VEGF in response to overexpression of ZNF24. The correlative data on inverse expression patterns of ZNF24 and VEGF from multiple systems suggested that this transcription factor may be involved in the negative regulation of Figure 2. ZNF24 and VEGF expression levels are inversely correlated in VEGF. To better determine the functional relationship between matched cDNA pairs of malignant and adjacent normal human tissues. ZNF24 and VEGF expression, MDA-MB-231 cells that stably A, up-regulated expression of ZNF24 was associated with decreased VEGF expression in four of the five colon pairs analyzed (samples 1, 3, 4, and 5). overexpress ZNF24 (MDA-MB-231-ZNF24) were established. These Additionally, ZNF24 was down-regulated in three of the tumor (T) samples cells and parental MDA-MB-231 cells were cultured under either compared with adjacent normal (N) tissues (samples 1, 3, and 4). B, ZNF24 was normoxic or hypoxic conditions for 24 h. To ascertain the effect of inversely correlated with VEGF expression in three of the five breast pairs analyzed (samples 1, 2, and 3).Strikingly, ZNF24 was significantly overexpression of ZNF24 on VEGF secretion, the levels of secreted down-regulated in all five breast carcinoma samples compared with normal VEGF in conditioned media from these cell lines were assayed mammary tissues.

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Taken together, these data suggest that ZNF24 may be involved in transcriptional repression of VEGF. Repression of genes, including VEGF, can occur via a number of mechanisms, including, but not limited to, repression of transcription, a decrease in mRNA stability, or premature degradation of the synthesized protein (22). We hypothesized that ZNF24 was func- tioning as a transcriptional repressor for a number of reasons. First, the C-terminal domain of ZNF24 contains four Kru¨ppel-like Cys2- His2 zinc finger motifs that encode putative DNA-binding domains (14). Second, ZNF24 was previously reported to possess transrepression activity against a GAL4 reporter plasmid in mammalian Chinese hamster ovary and NIH3T3 cells (14). Finally, another member of the SCAN domain–containing Cys2-His2 transcription factor family, ZNF174, has been characterized as a novel transcriptional repressor of both the platelet-derived growth factor-B and transforming growth factor-h promoters (23). Therefore, based on the secondary structure of ZNF24, the reported transcriptional repression by ZNF24 and the fact that ZNF24 and ZNF174 belong to the same transcription factor family, we designed our studies to determine whether ZNF24 is involved in repression of VEGF transcription. The effect of ZNF24 on VEGF transcription was investigated by analyzing the effect of overexpression of ZNF24 on the transcriptional activity of the VEGF promoter using VEGF promoter luciferase constructs. Overexpression of ZNF24 resulted in a significant decrease in luciferase activity, which corresponds to decreased VEGF promoter activity, supporting our hypothesis that ZNF24 may negatively regulate VEGF expression through transcriptional repression. Negative regulation of VEGF transcription by ZNF24 could occur via a number of mechanisms. For example, ZNF24 may bind the VEGF promoter directly either by itself, as part of a transcriptional repression complex, or indirectly as part of a complex that binds the promoter via one of the other molecules comprising that complex. The N-terminal region of ZNF24 contains a SCAN box domain, a leucine-rich domain that has been implicated as an oligomerization domain mediating protein-protein interactions to form homoligomers or heteroligomers with other proteins containing SCAN domains (24–26). Therefore, it is possible that ZNF24 Figure 3. Effects of overexpression or silencing of ZNF24 on VEGF expression, comprises part of a transcriptional repression complex that binds secretion, and promoter activity. A, VEGF protein levels were significantly decreased in multiple MDA-MB-231-ZNF24 clones compared with parental the VEGF promoter via the DNA-binding domains of ZNF24 and/ MDA-MB-231 cells.Down-regulation of VEGF was observed when cells were or other potential DNA-binding molecules within the complex. B, cultured under both normoxic or hypoxic conditions. transfection of MCF-7 Alternatively, ZNF24, either alone or as part of a complex, may bind cells with ZNF24 small interfering RNAs resulted in silencing of ZNF24 expression with a concomitant increase in VEGF expression.Transfection with the VEGF promoter at a site that hinders binding of transcriptional control small interfering RNAs had no effect on ZNF24 or VEGF expression. activators of VEGF, such as HIF-1 and Sp1. It is also possible that C, schematic representation of the VEGF promoter reporter construct.Eight kilobases of VEGF sequence upstream of the transcription initiation site through ZNF24 indirectly inhibits VEGF promoter activity by sequestering 890 bp of the 5¶ untranslated region was fused to the firefly luciferase gene (Luc) a transcriptional activator in a fashion similar to the inhibition of as a reporter for VEGF promoter activity (9kb-Luc).A promoter-less luciferase VEGF expression by the von Hippel Lindau gene product that gene (pGL2-Luc) was used as a negative control for promoter activity. D, overexpression of ZNF24 results in significant repression of VEGF promoter sequesters Sp1 and thus prevents it from activating VEGF tran- activity indicated by decreased luciferase activity.A CMV promoter–driven scription (9). Finally, ZNF24 may act as a transcriptional activator, luciferase was used as a positive control for promoter activity (data not shown). turning on the expression of a separate transcription factor, for example, which in turn is responsible for repression of VEGF. These potential mechanisms are currently being investigated in our Discussion laboratory. Whereas many positive regulators of VEGF expression have The data presented here show that VEGF expression is negatively been identified, relatively little is known regarding the negative regulated by ZNF24 or a ZNF24-mediated mechanism, suggesting a regulation of VEGF. In the present study, we identified a trans- tumor suppressor role for ZNF24. ZNF24 was down-regulated in cription factor, ZNF24, whose expression was inversely correlated 60% of the colon carcinomas and in 100% of the breast carcinomas with expression of VEGF in a number of experimental systems. that we analyzed compared with respective adjacent normal Moreover, silencing of ZNF24 expression resulted in up-regulation tissues. Down-regulation of ZNF24 expression in tumors is of VEGF whereas overexpression of ZNF24 resulted in both consistent with our hypothesis that ZNF24 may be involved in decreased VEGF expression and decreased VEGF promoter activity. negative regulation of tumor growth by repressing expression of www.aacrjournals.org 8739 Cancer Res 2007; 67: (18). September 15, 2007

VEGF. ZNF24 may be down-regulated to promote VEGF expression expression of VEGF seems to be hypoxia-independent. A compa- during tumor progression and other angiogenesis-dependent rable decrease in VEGF expression was observed whether MDA- diseases characterized by aberrant VEGF expression. Furthermore, MB-231-ZNF24 cells were cultured under normoxic or hypoxic ZNF24 is localized to a chromosomal locus (18q12) that is mutated conditions. Although VEGF expression was decreased regardless of in a number of human cancers, such as colorectal carcinomas oxygen conditions, the overall VEGF expression was still higher in (17, 18), head and neck cancer (27), invasive breast cancer (21), cells cultured under hypoxia compared with normoxic conditions, gastric cardia adenocarcinomas (28), testicular germ cell tumors suggesting that hypoxia-inducible expression of VEGF can still (29), and a number of hematopoietic malignancies (30). The high occur in MDA-MB-231-ZNF24 cells, albeit, at a significantly incidence of mutations within this chromosomal locus strongly decreased rate. suggests the presence of potential tumor suppressor genes within Overexpression of ZNF24 results in decreased expression of this region. To date, no mutations in ZNF24 have been associated VEGF with an associated decrease in VEGF promoter activity. with malignancies; however, given its negative effect on VEGF These data suggest that ZNF24 may represent a novel transcrip- expression, it would be informative to knockout ZNF24 expression tional repressor of VEGF and, as such, may be an addition to the or construct dominant negative ZNF24 mutations to determine relatively small number of negative regulators of VEGF expression the result on repression of VEGF and on subsequent angiogenesis that have been identified. Elucidating the mechanism and and/or tumor progression. consequence of VEGF repression by ZNF24 may be useful in the Whereas it seems that ZNF24 may be involved in the negative development of therapeutic approaches to treating angiogenesis- regulation of VEGF expression, it is unclear how expression of dependent pathologies by turning VEGF on or off, using ZNF24 as ZNF24 is regulated. The data presented here indicate that ZNF24 is the switch. Recently, it has been shown that gene therapy down-regulated during conditions in which HIF-1 is activated, for experiments using artificially designed zinc finger transcription example in preangiogenic tumor nodules and in cells cultured factors can activate expression of VEGF in vivo, leading to under hypoxia, suggesting that expression of ZNF24 might be stimulation of angiogenesis (31, 32). The potential exists for the regulated by hypoxia. Hypoxia induces increased VEGF expression development of similar technologies based on the inactivation of through activation or up-regulation of positive regulators of VEGF VEGF expression via ZNF24. expression, such as HIF-1 (8) and Ets-1 (31). However, it is also possible that negative regulators of VEGF expression, such as Acknowledgments ZNF24, could be down-regulated, augmenting the increase in VEGF Received 5/3/2007; accepted 6/28/2007. production. Elucidating the hypoxia-mediated and nonhypoxia- Grant support: NIH RO1 CA83106 (M.A. Moses), NIH PO1 CA45548 (M.A. Moses mediated mechanisms for regulating expression of ZNF24 would be and P.A. D’ Amore), and ACS RPG83821 (M.A. Moses). The costs of publication of this article were defrayed in part by the payment of page informative, both in terms of understanding the regulation of this charges. This article must therefore be hereby marked advertisement in accordance transcription factor and in characterizing potentially novel with 18 U.S.C. Section 1734 solely to indicate this fact. mechanisms involved in the negative regulation of VEGF. It is We thank Dr. Eric Ng for the design and development of the VEGF promoter reporter construct, Dr. Michael Manfredi for assay supports and Drs. Tucker Collins important to note that although expression of ZNF24 may be and Tara Sander of the Department of Pathology at Children’s Hospital, Boston, MA regulated by hypoxia, the mechanism by which ZNF24 represses for helpful discussions.

References 9. Mukhopadhyay D, Knebelmann B, Cohen HT, Ananth 16. Rousseau-Merck MF, Huebner K, Berger R, Thiesen HJ. S, Sukhatme VP. The von Hippel-Lindau tumor sup- Chromosomal localization of two human zinc finger pro- 1. Ferrara N, Carver-Moore K, Chen H, et al. Heterozy- pressor gene product interacts with Sp1 to repress tein genes, ZNF24 (KOX17) and ZNF29 (KOX26), to 18q12 gous embryonic lethality induced by targeted inactiva- vascular endothelial growth factor promoter activity. and 17p13–12, respectively. Genomics 1991;9:154–61. tion of the VEGF gene. Nature 1996;380:439–42. Mol Cell Biol 1997;17:5629–39. 17. Kern SE, Fearon ER, Tersmette KW, et al. Clinical and 2. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood 10. Cockman ME, Masson N, Mole DR, et al. Hypoxia pathological associations with allelic loss in colorectal vessel development and lethality in embryos lacking a inducible factor-a binding and ubiquitylation by the von carcinoma [corrected]. JAMA 1989;261:3099–103. single VEGF allele. Nature 1996;380:435–9. Hippel-Lindau tumor suppressor protein. J Biol Chem 18. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic 3. Klagsbrun M, D’Amore PA. Vascular endothelial 2000;275:25733–41. alterations during colorectal-tumor development. N Engl growth factor and its receptors. Cytokine Growth Factor 11. Pal S, Datta K, Mukhopadhyay D. Central role of p53 J Med 1988;319:525–32. Rev 1996;7:259–70. on regulation of vascular permeability factor/vascular 19. Fang J, Shing Y, Wiederschain D, et al. Matrix 4. Aiello LP, Pierce EA, Foley ED, et al. Suppression of endothelial growth factor (VPF/VEGF) expression in metalloproteinase-2 is required for the switch to the retinal neovascularization in vivo by inhibition of mammary carcinoma. Cancer Res 2001;61:6952–7. angiogenic phenotype in a tumor model. Proc Natl Acad vascular endothelial growth factor (VEGF) using soluble 12. Senoo M, Matsumura Y, Habu S. TAp63g (p51A) and Sci U S A 2000;97:3884–9. VEGF-receptor chimeric proteins. Proc Natl Acad Sci dNp63a (p73L), two major isoforms of the p63 gene, 20. Fang J, Yan L, Shing Y, Moses MA. HIF-1a-mediated U S A 1995;92:10457–61. exert opposite effects on the vascular endothelial up-regulation of vascular endothelial growth factor, 5. Ohno-Matsui K, Hirose A, Yamamoto S, et al. Inducible growth factor (VEGF) gene expression. Oncogene 2002; independent of basic fibroblast growth factor, is expression of vascular endothelial growth factor in adult 21:2455–65. important in the switch to the angiogenic phenotype mice causes severe proliferative retinopathy and retinal 13. Zhang L, Yu D, Hu M, et al. Wild-type p53 suppresses during early tumorigenesis. Cancer Res 2001;61:5731–5. detachment. Am J Pathol 2002;160:711–9. angiogenesis in human leiomyosarcoma and synovial 21. Richard F, Pacyna-Gengelbach M, Schluns K, et al. 6. Loureiro RM, Maharaj AS, Dankort D, Muller WJ, sarcoma by transcriptional suppression of vascular Patterns of chromosomal imbalances in invasive breast D’Amore PA. ErbB2 overexpression in mammary cells endothelial growth factor expression. Cancer Res 2000; cancer. Int J Cancer 2000;89:305–10. upregulates VEGF through the core promoter. Biochem 60:3655–61. 22. Loureiro RM, D’Amore PA. Transcriptional regulation Biophys Res Commun 2005;326:455–65. 14. Han ZG, Zhang QH, Ye M, et al. Molecular cloning of of vascular endothelial growth factor in cancer. 7. Pal S, Claffey KP, Cohen HT, Mukhopadhyay D. six novel Kruppel-like zinc finger genes from hemato- Cytokine Growth Factor Rev 2005;16:77–89. Activation of Sp1-mediated vascular permeability fac- poietic cells and identification of a novel transregulatory 23. Williams AJ, Khachigian LM, Shows T, Collins T. tor/vascular endothelial growth factor transcription domain KRNB. J Biol Chem 1999;274:35741–8. Isolation and characterization of a novel zinc-finger requires specific interaction with protein kinase C ~. 15. Shi SL, Liu MM, Yu L, et al. [Assignment of a novel protein with transcription repressor activity. J Biol J Biol Chem 1998;273:26277–80. zinc finger gene ZNF191 to human chromosome Chem 1995;270:22143–52. 8. Semenza GL. HIF-1: using two hands to flip the 18Q12.1 by human/rodent somatic cell hybrid panel 24. Schumacher C, Wang H, Honer C, et al. The SCAN angiogenic switch. Cancer Metastasis Rev 2000;19: and fluorescent in situ hybridization]. Shi Yan Sheng Wu domain mediates selective oligomerization. J Biol Chem 59–65. Xue Bao 1998;31:21–7. 2000;275:17173–9.

Cancer Res 2007; 67: (18). September 15, 2007 8740 www.aacrjournals.org

25. Stone JR, Maki JL, Blacklow SC, Collins T. The SCAN 28. van Dekken H, Alers JC, Riegman PH, et al. Molecular activation to oncogenic growth transformation. J domain of ZNF174 is a dimer. J Biol Chem 2002;277: cytogenetic evaluation of gastric cardia adenocarcinoma Immunol 2002;168:680–8. 5448–52. and precursor lesions. Am J Pathol 2001;158:1961–7. 31. Liu PQ, Rebar EJ, Zhang L, et al. Regulation of an 26. Williams AJ, Blacklow SC, Collins T. The zinc finger- 29. Skotheim RI, Kraggerud SM, Fossa SD, et al. Familial/ endogenous locus using a panel of designed zinc finger associated SCAN box is a conserved oligomerization bilateral and sporadic testicular germ cell tumors show proteins targeted to accessible chromatin regions. Acti- domain. Mol Cell Biol 1999;19:8526–35. frequent genetic changes at loci with suggestive linkage vation of vascular endothelial growth factor A. J Biol 27. Papadimitrakopoulou VA, Oh Y, El-Naggar A, et al. evidence. Neoplasia 2001;3:196–203. Chem 2001;276:11323–34. Presence of multiple incontiguous deleted regions at the 30. Tune CE, Pilon M, Saiki Y, Dosch HM. Sustained 32. Rebar EJ, Huang Y, Hickey R, et al. Induction of angio- long arm of chromosome 18 in head and neck cancer. expression of the novel EBV-induced zinc finger gene, genesis in a mouse model using engineered transcription Clin Cancer Res 1998;4:539–44. ZNFEB, is critical for the transition of B lymphocyte factors. Nat Med 2002;8:1427–32.

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Cancer Res 2007;67:8736-8741.

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