Oncogenes and tumor angiogenesis - Nature

Oncogene (2000) 19, 4611 ± 4620 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc

Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner Omar LoÂpez-Ocejo1,7, Alicia Viloria-Petit3,7, MoÂnica Bequet-Romero2, Debabrata Mukhopadhyay5, Janusz Rak6 and Robert S Kerbel*,3,4 1

Vaccine Division, Centre for Genetic Engineering and Biotechnology, PO Box 6162, C Havana, 10600, Cuba; 2Pharmaceutical Division, Centre for Genetic Engineering and Biotechnology, PO Box 6162, C Havana, 10600, Cuba; 3Division of Cancer Biology Research, Sunnybrook and Women's College Health Sciences Centre, S-218, 2075 Bayview Ave, Toronto, Ontario M4N 3M5, Canada; 4Department of Medical Biophysics, University of Toronto, Division of Cancer Biology Research, Sunnybrook and Women's College Health Sciences Centre, S-218, 2075 Bayview Ave, Toronto, Ontario M4N 3M5, Canada; 5Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, MA 02215, USA; 6 Hamilton Civic Hospitals Research Centre, McMaster University, 711 Concession Street, Hamilton, Ontario, L8W 1C3, Canada

Like other types of pre-malignant lesions and carcinoma, angiogenesis is associated with high-grade cervical dysplasia and with invasive squamous carcinoma of the cervix. Vascular endothelial cell growth factor (VEGF) is known to be one of the most important inducers of angiogenesis and is upregulated in carcinoma of the cervix. Human Papilloma Virus 16 (HPV-16) has been etiologically linked to human cervical cancer, and the major oncogenic proteins encoded by the viral genome, E6 and E7, are involved in the immortalization of target cells. Because several oncogenes including mutant ras, EGF receptor, ErbB2/Her2, c-myc and v-src upregulate VEGF expression, we asked whether HVP-16 E6 oncoprotein could act in a similar fashion. We found that HPV-16 E6-positive cells generally express high levels of VEGF message. Furthermore, co-expression of the VEGF promoter-Luc (luciferase) reporter gene with E6 in both human keratinocytes and mouse ®broblast showed that E6 oncoprotein upregulates VEGF promoter activity, and does so in a p53 independent manner. An E6 responsive region which comprises four Sp-1 sites, between 7194 and 750 bp of the VEGF promoter, is also necessary for constitutive VEGF transcription. Taken together, our results suggest the possibility that the HPV oncoprotein E6 may contribute to tumor angiogenesis by direct stimulation of the VEGF gene. Oncogene (2000) 19, 4611 ± 4620. Keywords: tumor; angiogenesis; oncogenes; cervical cancer Introduction It is now well established that angiogenesis, a process of recruitment of new blood vessel capillaries from a preexisting network of mature vessels, is a fundamental prerequisite for progressive expansion of tumors. The ability of tumors to induce and sustain angiogenesis is thought to be the consequence of two major types of functional change. The ®rst is a gain-of-function event

*Correspondence: RS Kerbel 7 These authors contributed equally Received 20 March 2000; revised 17 July 2000; accepted 24 July 2000

in which growth factors acting as stimulators of angiogenesis are induced or upregulated (Hanahan and Folkman, 1996; Hanahan, 1997). Examples of such stimulators include basic ®broblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF) ± also known as vascular permeability factor (VPF) ± and angiopoietin-1, among a number of others (Klagsbrun and Soker, 1993; Bouck et al., 1996; Davis and Yancopoulos, 1999). The second change is a lossof-function event, namely, the loss, or downregulation of endogenous protein inhibitors such as thrombospondin-1 and interferon a (Bouck et al., 1996). Our laboratory has been studying the means by which tumors induce or upregulate proangiogenic growth factors, especially VEGF. In this regard, it is well known that hypoxia, a common environmental feature of solid tumors, is an inducer of VEGF expression (Ferrara, 1995). In addition, we have been analyzing various oncogenes as possible inducers of VEGF and hence, by extension, as contributory factors in tumor angiogenesis (Rak et al., 1995a,b). For example, induction of VEGF appears to be a consequence of oncogenic ras mutations both in vitro and in vivo (Kerbel et al., 1998). This relationship may be necessary, but not sucient, for the angiogenic and tumorigenic phenotype (Okada et al., 1998; Chin et al., 1999). Other oncogenes which appear to have a similar eect on VEGF expression include receptor tyrosine kinases such as the ErbB family including the EGF receptor and ErbB2/Her2 (Viloria-Petit et al., 1997), non-receptor kinases such as src (Mukhopadhyay et al., 1995a; Rak et al., 1995a,b; Ellis et al., 1998) and transcription factor encoding proto-oncogenes such as myc, c-fos, and c-jun (Saez et al., 1995; Kraemer et al., 1999; Pelengaris et al., 1999). In this regard one important type of oncogene, which has not yet been examined well for its impact on VEGF expression, and hence tumor angiogenesis, are those encoded by the human papilloma virus (HPV). The human papilloma viruses (HPV) are a group of epitheliotropic small DNA viruses that have been strongly linked to the etiology of human anogenital cancer, particularly cervical cancer (Mansur and Androphy, 1993; Zur-Hausen and de Villiers, 1994; Stoler, 2000). Indeed, 99.7% of invasive cervical carcinomas worldwide contain and express DNA from HPV (Walboomers et al., 1999; Herrington, 1999), with

HPV-16 E6 oncoprotein and VEGF in tumor angiogenesis O LoÂpez-Ocejo et al

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HPV-16 and 18 being the types most frequently found in these tumors. The early viral proteins E6 and E7 are continually expressed in HPV-associated cervical cancer tissues and cell lines derived from cervical tumors (Hawley-Nelson et al., 1989; Androphy et al., 1987; Choo et al., 1987; Schwarz et al., 1985). These two oncoproteins can physically interact with and functionally disrupt two major cell cycle regulatory proteins, namely p53 and the retinoblastoma gene product (pRb), respectively (Werness et al., 1990; Dyson et al., 1989). Inactivation of tumor suppressor proteins by these interactions helps explain why transfection of normal human exocervical and cervical epithelial cells ± the major in vivo target cells for HPV infection ± with cloned HVP-16 and HVP-18 DNA can result in cell transformation (Barbosa and Schlegel, 1989; Munger et al., 1989; Woodworth et al., 1988). In this respect the interaction of E6 with p53 in vitro promotes rapid degradation of p53 via interaction with E6AP protein, a component of the ubiquitin pathway (Schener et al., 1993; Song et al., 1998). In vivo experiments using mice transgenic for HPV16 oncogenes have shown that either E6 or E7 alone is sucient to induce tumor formation (Coussens et al., 1996; Smith-McCune et al., 1997; Song et al., 1999). Recent studies indicate that E6 induces transcription of genes involved in cell growth and transformation such as c-fos, c-myc and cytokines such as TGFb1 (Morosov et al., 1994; Kinoshita et al., 1997; Dey et al., 1997). A similar relationship may also hold true for regulators of angiogenesis such as VEGF. With respect to HPVassociated cancers, high grade cervical dysplasia and invasive squamous cell carcinoma of the cervix are, like many other types of cancer, associated with angiogenesis (Smith-McCune and Weidner, 1994; Guidi et al., 1995; Sillman et al., 1981; Smith-McCune et al., 1997; Hove et al., 1999; Tokumo et al., 1998). However the mechanisms responsible for induction and maintenance of the angiogenic phenotype in cervical neoplasia are unknown. Given the relationship of oncogenes and angiogenesis in other types of tumors or experimental systems, we hypothesized that part of the transforming capacity of E6 and E7 oncogenes would likely encompass upregulation of pro-angiogenic activities required for expression of the tumorigenic phenotype in vivo. Among known pro-angiogenic factors, VEGF has been shown to be consistently upregulated in cervical neoplasia (Neufeld et al., 1999; Hove et al., 1999; Tokumo et al., 1998). In addition, studies with keratin-14-HPV-16 transgenic mice expressing E6-E7 (Smith-McCune et al., 1997) as well as with tissue samples of human cervix, suggest that both HPV oncogenes and hypoxia may act as mediators of VEGF upregulation (Coussens et al., 1996; SmithMcCune et al., 1997). A contribution of the HPV oncoproteins to VEGF upregulation is also suggested by the similar pattern of incremental upregulation of both the HPV oncogenes and VEGF mRNA as well as protein detected in dierent in vitro and in vivo multistep carcinogenesis models involving transgenic `oncomice' (Coussens et al., 1996; Park et al., 1995; Smith-McCune et al., 1997). The purpose of the present work was to study the mechanisms involved in VEGF upregulation in HPV positive cells. By using transient transfection assays, we

show that the transcriptional activity of the VEGF promoter is induced by the HPV-16 E6 oncoprotein and that this induction is p53-independent. Our results suggest the possibility that the E6 oncogene may contribute to the growth and development of cervical tumors in vivo by promoting VEGF-dependent angiogenesis.

Results Upregulation of endogenous VEGF mRNA expression and protein secretion in HPV-16-positive cells As an initial step to study the angiogenic potential of HPV-16-positive cells, we compared the levels of expression of VEGF mRNA in both immortalized human keratinocytes, which represents the host cell for HPV infection, and cervical cancer- derived cells, including the HPV-negative cell line C33A and two HPV-16-positive cell lines (HeLa and CaSki) by Northern blotting. VEGF mRNA levels were increased by twofold in the HPV-negative cervical cancer cell line C33A compared to the human immortalized keratinocytes HaCaT (Figure 1a). However, VEGF mRNA levels were higher (4 ± 7-fold greater than the level found in HaCaT cells) in the HPV-16-positive cells, including the cervical cancer-derived cell lines HeLa and CaSki, as well as the positive control cell line HPK1A, infected with HPV-16 genome (Figure 1a). In addition, HeLa cells showed an increase of 8 ± 18-fold in their levels of VEGF mRNA as compared to HaCaT keratinocytes under increasing serum concentrations (Figure 1b). In agreement with the Northern analysis, VEGF secretion by HaCaT cells was virtually undetectable in low serum conditioned medium (0.1% FBS), whereas HeLa cells secreted up to 200-fold more immunodetectable VEGF under the same conditions (Figure 1c). VEGF upregulation by HeLa cells does not involve the TGF a/EGFR autocrine loop The involvement of HPV in the development of carcinomas of the uterine cervix has been well established (Walboomers et al., 1999). However, other genetic alterations may contribute to the pathogenesis of cervical cancer (Lazo, 1999). In this regard, most of the HPV-associated dysplasias and cervical carcinomas also express high levels of the EGFR as well as EGFR ligands, such as TGF-a (Derynck et al., 1987; Ben-Bassat et al., 1997; Kersemaekers et al., 1999). Moreover, HeLa cells have been shown to posses high expression of EGFR, which appears to be driven by their intrinsic expression of the HPV proteins E6 and E7 (Hu et al., 1997). In addition, activation of the EGFR (growth factor/receptor) system has been shown to upregulate VEGF expression (Viloria-Petit et al., 1997; Detmar et al., 1994; Goldman et al., 1993; Gille et al., 1997). Therefore we hypothesized that in HPV positive cells upregulation of VEGF may be EGFR-mediated. To investigate whether or not this is the case we studied the eect of anti-EGFR (C225 and hR3) and antiTGF-a (Ab-3) monoclonal neutralizing antibodies on the levels of VEGF protein produced by HeLa cells.


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Figure 1 Comparison of VEGF mRNA and protein levels in HPV-16- positive and HPV-16- negative cells. (a) Northern blot analysis of VEGF expression in a panel of cell lines including human immortalized HPV-16- negative (HaCaT) and HPV-16positive (HPK1A) keratinocytes, as well as cervical carcinoma derived HPV-16- negative (C33A) and HPV-16- positive (HeLa, CaSki) cells. VEGF mRNA levels were increased 4 ± 7-fold in HPV-16- positive cells as compared to HPV-16- negative immortalized keratinocytes, and 2 ± 3-fold compared to the HPV-16- negative cervical carcinoma cell line C33A. (b) Northern blot analysis of VEGF expression in HeLa cells as compared to HaCaT keratinocytes. VEGF mRNA levels were increased 7 ± 18-fold in HeLa, with the maximum dierence observed at low serum concentration (0.1%). The 28S and 18S mRNA bands show equal loading and RNA integrity (lower panel). Fold activation was calculated by densitometry with 28S serving as a normalization control. The value obtained for HaCaT keratinocytes in 0.1% FBS was taken as the basal level (1.0) in (b). (c) ELISA assay results showing the increase in VEGF concentration in conditioned medium collected from HeLa cells compared to that from HaCaT keratinocytes. Cells were incubated for 24 h after being seeded at dierent serum concentrations Oncogene


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High concentrations of either anti-EGFR or the antiTGFa neutralizing antibodies were unable to cause any signi®cant downregulation of the VEGF protein levels secreted into conditioned medium (Figure 2a,c). In contrast, both EGFR-neutralizing antibodies (C225 and hR3) showed a remarkable eect on the levels of VEGF protein secreted by the human squamous cell carcinoma cell line A431 (Figure 2b), a cell system where VEGF expressions appears to be highly dependent on EGFR activity (Viloria-Petit et al., 1997). These results suggest that, at least in HeLa cells, VEGF upregulation does not appear to occur via an endogenous mechanism involving an EGFR autocrine loop. HPV-16 E6 oncoprotein activates the VEGF promoter Given the potential of HPV-16 encoded oncoproteins to function as transcription factors (Morosov et al.,

1994; Kinoshita et al., 1997; Dey et al., 1997), we hypothesized that the HPV-16 E6 oncoprotein may more directly trigger VEGF gene expression. We reasoned that the mechanism of E6 dependent upregulation of VEGF may be deduced from an analysis of the ability of this oncogene to activate the VEGF promoter. For this purpose, the reporter construct 2.6 VEGF was transfected into the human immortalized keratinocytes HaCaT and NIH3T3 ®broblasts together with the HPV-16 E6 expression plasmid (pJ4O16E6) or a control plasmid (pJ4O). As shown in Figure 3b, transient expression of the E6 protein in HaCaT cells results in a dose-dependent (up to 2 ± 2.5-fold) increase of VEGF promoter activity as compared to vector control. This induction does not appear to be cell-speci®c, since an equivalent response was observed after transient transfection of NIH3T3 mouse ®broblasts (Figure 3c).

Figure 2 Eect of blockade of EGFR signaling on the production of VEGF protein by HeLa and A431 cells. (a) Eect of treatment with two anti-EGFR antibodies, C225 and hR3, on the secretion of VEGF by Hela cells. (b) Eect of equal treatments on the production of VEGF protein by A431 cells. No signi®cant dierences among the distinct treatment culture conditions in terms of VEGF secretion were detected in HeLa cells. In contrast, both antibodies down-regulated VEGF protein levels signi®cantly, up to a maximum of 50%, in A431 cells. (c) In agreement with the lack of eect of the EGFR neutralizing antibodies on HeLa, the anti-TGF-a antibody Ab-3 also failed to down-regulate VEGF protein production by HeLa cells. A one-way ANOVA test was used to compare the dierent treatment culture conditions for each experiment. A Dunnet post-test was used in order to compare each result with its respective control Oncogene


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Figure 3 Transcriptional activation of VEGF promoter by a plasmid expressing the HPV-16 E6 oncoprotein. (a) Schematic representation of the activator and the reporter construct. The plasmid pJ4O16E6 expresses the E6 gene of HPV-16, driven by MoMu LV LTR promoter (Crook et al., 1991). The reporter 2.6 VEGF is a VEGF promoter (72361 to +298)-Luciferase construct described previously (Mukhopadhyay et al., 1997). (b and c) Eect of HPV-16 E6 gene product on VEGF promoter activity in either HaCaT keratinocytes or NIH3T3 cells, respectively. Signi®cant dierences (P50.01) were detected for both cell lines by applying unpaired t-test. Dose-dependence of VEGF-promoter induction by E6 expression is seen in (b). Plasmid 2.6 VEGF (1.5 mg) was co-transfected into HaCaT cells along with increasing concentrations of either HPV-16 E6 (0 ± 3 mg) expressing plasmid or control plasmid pJ4O (3 mg). Values are presented as averages of three independent experiments in terms of normalized luciferase activity with the standard error (s.e.) of the mean noted. Fold induction is indicated at the top of the bars

HPV-16 E6-mediated transactivation of VEGF promoter involves region 7194 to 750 In order to localize the region involved in the E6mediated transactivation, a series of 5' deletion constructs of the 2.6 VEGF promoter-reporter vector were co-transfected with the pJ4O16E6 expression plasmid or the empty expression vector pJ4O into both human immortalized keratinocytes HaCaT and NIH3T3 mouse embryo ®broblasts (Figure 4). An approximated twofold stimulation of the promoter activity was observed with two dierent deletion constructs: 1.5 VEGF (bp 71226 to +298) and 0.35 VEGF (bp 7194 to +157). In contrast, the 0.2 VEGF (bp 750 to +157) construct, which lacks four

consensus Sp-1 sites, showed loss of both the basal and the E6 mediated promoter activity. Similar results were observed when the same promoter analysis was performed in NIH3T3 cells (data not shown). The HPV-16 E6-mediated transactivation of the VEGF promoter is p53-independent It is generally assumed that the transcribing capacity of HPV-16 oncogenes is related to their interference with the function of p53 and the Rb gene products. In addition, p53 has been shown to act as a suppressor of VEGF expression (Kieser et al., 1994), including at the transcriptional level (Mukhopadhyay et al., 1995b). It was therefore surprising to ®nd that four proximal SpOncogene


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Figure 4 Identi®cation of the E6-responsive region in the human VEGF promoter. Dierent deletion constructs were cotransfected into HaCaT cells with either E6 expression plasmid (&) or control plasmid (&). Left panel presents a schematic representation of the VEGF promoter deletions including consensus binding sites of dierent transcription factors. The E6responsive region is localized between bp 7194 and 750 of the VEGF gene promoter. Values are presented as averages of three independent experiments in function of normalized luciferase activity with the s.e. of the mean noted. Fold induction in luciferase activity is depicted to the right side of the bars

1 consensus sites of the VEGF 5' UTR suce to explain the apparent impact of E6 in promoter activity. However, it is also known that Sp-1 DNA binding activity is in¯uenced by interactions with p53 (Webster et al., 1996; Bargonetti et al., 1997); hence, VEGF expression may ultimately be controlled by such mechanisms. In order to determine whether this is indeed the case we decided to evaluate the eect of HPV-16 E6 on VEGF promoter activity using mouse embryonic ®broblasts (MEF), lacking both copies of the p53 gene (p537/7), as recipient cells. As shown in Figure 5, E6 was still able to cause a twofold induction of VEGF promoter activity in the absence of p53, suggesting that transactivation of the VEGF promoter by HPV-16 E6 is independent of its ability to inactivate p53. Discussion The rationale for undertaking the studies reported here was based on four main considerations. First, VEGF is considered to be of paramount importance in tumor angiogenesis (Klagsbrun and Soker, 1993; Ferrara, 1995; Hanahan and Folkman, 1996; Hanahan, 1997). Second, included among the many inducers of VEGF in tumors are a variety of oncogenes, such as mutant ras, activated src, and activated erbB2/Her-2 and EGF receptor tyrosine kinases (Rak et al., 1995a,b; Kerbel et al., 1998), suggesting that a signi®cant generic function of oncogenes is to contribute to the angiogenic competence of tumors (Rak et al., 1995a,b; Kerbel et al., 1998). Third, HPV-16-encoded E6 and E7 viral oncoproteins have been implicated in the etiology of cervical cancer (Walboomers et al., 1999), as summarized in the introduction. Fourth, VEGF overexpression appears to be an important component of the angiogenic process in invasive squamous cell carcinoma of the cervix (Guidi et al., 1995; Sillman et al., 1981), similar to most other types of tumors. Increased VEGF expression and tumor vascularity have been observed in premalignant carcinoma of the cervix as well as high Oncogene

Figure 5 Activation of VEGF promotor by E6 in p53 null cells. The plasmid 2.6 VEGF (1.5 mg) was co-transfected into MEF p53+/+ or MEF p537/7 cells together with 3 mg of the HPV16 E6 expression plasmid (pJ4O16E6) or 3 mg of the control plasmid (pJ4O). The HPV-16 E6 appears to induce transcription from VEGF promoter even in the absence of a functional p53 gene. Values are presented as averages of three independent experiments in terms of normalized luciferase activity with the s.e. of the mean noted. Fold induction is indicated at the top of the bars

grade intraepithelial lesions and invasive carcinomas per se (Smith-McCune and Weidner, 1994, 1997; Guidi et al., 1995; Tokumo et al., 1998) Given this information, we reasoned that HPV associated oncoproteins, particularly E6, may be responsible, at least in part, for VEGF production in various stage lesions of carcinoma of the cervix. Consistent with this hypothesis we found that cervix carcinoma-derived cells which were positive for HPV16 E6 expressed higher levels of VEGF mRNA (2 ± 3fold increase) than HPV-16 E6- negative cells. This dierence in VEGF expression was even more remarkable when the transformed HPV-16 E6-positive cells were compared to human immortalized keratino-


cytes, in which case a 4 ± 7-fold increase in VEGF mRNA was observed. In agreement with these ®ndings HPV-16 E6-positive HeLa cells expressed highly elevated levels of both VEGF mRNA (8 ± 18-fold increase) and secreted protein (up to 200-fold), compared to control human immortalized keratinocytes. Clearly such dierences could be explained by genetic alterations in addition to or other than the HPV-16 E6 oncogene status in the cells tested. Other genetic changes might include additional virus-encoded products (such as E7) as well as other cellular oncoproteins. Regarding HeLa cells, we examined the possible contribution of the TGFa/EGF receptor signaling system due to its demonstrated over-activity in HeLa cells (Derynck et al., 1987; Hu et al., 1997) and also because EGFR activation can trigger VEGF expression (Viloria-Petit et al., 1997; Detmar et al., 1994; Goldman et al., 1993; Gille et al., 1997). However, using speci®c blocking antibodies to both TGFa and EGF receptor we showed that this receptorligand system is not involved in VEGF expression in HeLa cells. These results could be explained if the EGFR signaling system had a minor role in VEGF expression in the context of a more profound eect of the HPV-16 oncoproteins, E6 and E7. The impact of EGFR on VEGF protein produced in HeLa cells could be diminished as a consquence of the fact that this stimulation is driven by Sp-1 activity (Gille et al., 1997). Since our present study suggests that HPV-16 E6 stimulates VEGF promoter activity through Sp-1 sites (Figure 4), it is possible that a common pathway requiring Sp-1 is already active in HPV-16-infected cells, making them refractory to further EGFRdependent stimulation. We therefore addressed the question of whether HPV-16-encoded oncoproteins themselves might trigger VEGF gene expression in a direct manner, given the fact that HPV major oncoproteins are known to have selective transcription regulatory properties. In particular, E6 has been demonstrated to transactivate several homologous and heterologous promoters including viral enhancers (Morosov et al., 1994; Kinoshita et al., 1997; Dey et al., 1997). We observed that E6 can indeed activate transcription from a VEGF promoter by a factor of two, and in a dosedependent manner in both HaCaT human immortalized keratinocytes and NIH3T3 ®broblasts (Figure 3b,c). There is no obvious explanation as to whether and how a twofold change in VEGF promoter activity can translate into the high levels of VEGF message found in the HPV-16-positive cell lines initially examined. One possibility could be that both HPVencoded oncoproteins, E6 and E7, impact VEGF expression and do so, not only by stimulating the activity of the promoter, but also by aecting mRNA stability. Indeed, other oncogenes (White et al., 1997) as well as the VHL tumor suppressor gene (Gnarra et al., 1996; Levy et al., 1996) have been found to increase the stability of VEGF mRNA. Additionally, other genetic changes present in the cervical cancer cells tested in the present study, may also have a positive impact on the overall level of VEGF expression. The former rather than the latter possibility is suggested by the high VEGF message levels found in HPK1A cells, which have HPV-16 genome as their predominant genetic alteration (Figure 1a). On

the other hand, even a twofold change in the activity of the VEGF promoter may in itself have biological relevance. For example, 2 ± 3-fold reductions in VEGF expression in tumor cells have been shown to produce surprisingly profound suppressive eects on tumor angiogenesis and tumor growth in vivo (Ferrara et al., 1996; Cheng et al., 1996; Okada et al., 1998; Kerbel et al., 1998). In HPV-16-positive cells where other genetic and epigenetic changes (e.g. hypoxia) could obviously increase VEGF expression, the E6 oncoprotein might contribute to amplifying this eect. Consistent with this hypothesis is recent evidence which suggests that the HPV-16 E6 oncoprotein may also contribute to the increase in VEGF message by interfering with the ubiquitin-mediated degradation of the hypoxia inducible factor-1 alpha (HIF-1a) (Ravi et al., 2000), a transcription factor involved in the activation of VEGF gene promoter in response to hypoxia (Semenza, 1999). Another important ®nding from our studies is that E6-mediated VEGF induction appears independent of the p53 gene. This ®nding is particularly surprising given the existing evidence of the p53-mediated repression of VEGF promoter (Mukhopadhyay et al., 1995b; Keiser et al., 1994) and the demonstrated role of HPV-16 E6 protein in targeting wildtype p53 ubiquitination and degradation (Schener et al., 1993; Crook et al., 1991). Nevertheless, our results suggest that HPV-16 E6 stimulatory activity on VEGF promoter might predominantly result from its positive impact on the activity of other transcription factors, such as Sp-1. As shown in Figure 4, the target region involved in the E6-mediated transactivation of the VEGF promoter was mapped between 7194 and 750, relative to the transcription initiation site. In this regard, the VEGF promoter is not known to possess a TATA box sequence, and it is precisely within this 150-nucleotide region that a cluster of Sp1 binding sites have been identi®ed (Tischer et al., 1991). In the absence of a TATA box, mechanisms other than direct recruitment of TATA-binding proteins have been implicated in positioning of the basal transcription complex and initiation of transcription from a de®nite site. Sp-1 transcription factors have been shown to function as crucial proteins in accurate transcription initiation from the so-called TATA-less promoters (Azizkhan et al., 1993). These observations appear to apply to the VEGF promoter as well, since our studies show that deletion of the region between 7194/750, which contains the Sp-1 binding sites, appears to be critical for both constitutive and E6 mediated activation of the VEGF promoter. Although p53 has been shown to interact with Sp-1, acting as a transcriptional repressor of Sp-1 activity (Webster et al., 1996; Bargonetti et al., 1997), we believe that HPV-16 E6 can promote Sp-1 transactivation of VEGF promoter in a p53 independent manner, since the same twofold increase of VEGF promoter activity was observed in cells with or without expression of wild type p53. This possibility is supported by previous ®ndings showing Sp-1-mediated activation of other promoters, such as p21, in a p53independent fashion (Nakano et al., 1997). Taken together, the results suggest that the E6 oncoprotein may act as a transactivator of VEGF promoter

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activity. This function could contribute to its role in oncogenic transformation in vivo, given the importance of VEGF in tumor angiogenesis (Ferrara, 1995; Hanahan and Folkman, 1996; Okada et al., 1998) and the critical impact of angiogenesis on tumor growth in vivo (Hanahan and Folkman, 1996; Bouck et al., 1996). The mechanisms underlying the stimulatory eect of E6 on VEGF promoter are not yet clear. Various elements responsible for E6 transactivation of genes have been proposed, e.g. the GC and CAAT elements in the herpes simplex virus thymidine kinase gene or cmyc promoter, NF-kB elements in the HIV LTR promoter, and Sp-1 elements in TGF-b1 promoter (Kinoshita et al., 1997; Dey et al., 1997; Desaintes et al., 1992). However, a direct involvement of E6 as a transcription factor working alone has not been documented, although it has been demonstrated that E6 binds to DNA in a nonselective manner (Mallon et al., 1987). It is possible that E6 transactivation is mediated through the interaction with pre-existing cellular factors that are involved in activation of certain promoter/enhancer elements. The deletion studies performed in this work point to the Sp-1 transcription factor as the major element for E6 mediated induction of VEGF promoter. The E6 oncogene as other viral oncoproteins such as v-Rel, v-Src, v-Ras, and v-Raf might aect the synthesis, DNA-binding activity or the transcriptional activating functions of cellular transcription factor Sp-1 (Sif et al., 1993; Miltenberger et al., 1995). In summary our ®ndings, taken together, suggest the following: (i) HPV-16 E6 positivity correlates with elevated levels of VEGF expression; (ii) VEGF induction in HeLa cells is independent of EGFR and its ligand (TGFa) activity; (iii) the E6 oncoprotein induces transcription from the VEGF promoter in a p53 independent manner; and (iv) elimination of the region localized between position 7194 to 750 of the VEGF promoter abrogates E6 responsiveness. These results provide the ®rst indication that E6 viral oncoprotein induces VEGF gene expression by promoter transcriptional activation. Direct induction of VEGF expression by HPV-16 E6 oncoprotein could co-operate with other genetic alterations and/or microenvironmental changes such as hypoxia (Ravi et al., 2000) to result in maximal elevations of VEGF expression, similar to the co-operative eects of hypoxia and other oncogenes, or combinations of activated oncogenes and mutant suppressor genes (Volpert et al., 1997) in inducing maximal upregulation of VEGF expression (Mazure et al., 1996). Thus blockade of E6 expression or function could conceivably result in indirect suppression of cervical cancer growth in vivo through a mechanism involving downregulation of VEGF-mediated angiogenesis.

®broblasts were cultured at 378C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum (GIBCO/BRL). HaCaT is a stable cell line established from human cutaneous keratinocytes spontaneously immortalized after repeated passages in vitro (Boukamp et al., 1988). C33A is a HPVnegative cervical cancer cell line, while HeLa and CaSki are both HPV-16-positive. HPK1A cells have been previously described (Rohlfs et al., 1991; Durst et al., 1987) and were generated by exogenous expression of HPV-16 DNA into human primary foreskin keratinocytes. All cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) with the exception of MEF cells, which were a generous gift from Dr Larry Donehower, and the HPK1A cell line, which was kindly provided by Dr Federico DeMarco. Antibodies The neutralizing anti-human EGFR monoclonal antibody C225 (Fan et al., 1993; Goldstein et al., 1995) and the monoclonal antibody hR3, directed against the same target (Mateo et al., 1997) were provided by ImClone Systems (New York, NY, USA) and the Centre of Molecular Immunology (CIM) (Havana, Cuba), respectively. The anti-TGF-a monoclonal antibody Ab-3 was purchased from Calbiochem (Cambridge, MA, USA). RNA isolation and Northern blot analysis Total RNA was isolated from cultured cells by using the Trizol reagent (GIBCO/BRL, Grand Island, NY, USA), essentially as described by the manufacturer. The RNA was resolved on a 1% agarose gel containing 6.6 mol/l formaldehyde and transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech, Pisscataway, NJ, USA). The blots were hybridized with a 32P-labeled 200 bp human VEGF sequence common for all known VEGF isoforms (a gift from Dr Harold Dvorak). The gel was stained with ethidium bromide to indicate equal loading of RNA from each sample. Levels of mRNA were quantitated by densitometry analysis using Scanlet Jet Plus and the Molecular Analyst software (Bio Rad, Hercules, CA, USA). Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions. Brie¯y, cells were plated at a density of 105 cells/0.5 ml/well in a 24 well plate, 24 h later the growth medium was replaced with fresh assay medium containing either EGFR-neutralizing-antibody (C225 or hR3) or anti-TGF-a monoclonal antibody Ab-3 (only for HeLa treatment) in DMEM supplemented with 1% FBS. Conditioned medium was collected after 24 h, cellular debris removed by centrifugation, and the medium was kept at 7708C until VEGF quantitation was undertaken. Cell number was determined immediately after medium recovery using a Coulter Counter ZM (Coulter Electronics, Luton, UK). Plasmids and DNA transfection

Material and methods Cell lines and culture conditions The human cervical carcinoma cell lines C33A, HeLa and CaSki; human immortalized keratinocytes HaCaT, and HPK1A; the human vulvar squamous carcinoma A431; as well as NIH3T3, MEF p537/7 and MEF p53+/+ mouse Oncogene

The human VEGF promoter constructs 2.6 VEGF (bp 72371 to +298) and deletion mutants 1.5 VEGF (bp 71226 to +298), 0.35 VEGF (bp 7194 to +157) and 0.2 VEGF (bp 750 to +157) have previously been described (Mukhopadhyay et al., 1997). These constructs contain the fragments of the VEGF promoter coupled to ®re¯y luciferase. The E6-expressing construct pJ4OE6 and the empty vector used as control: pJ4O were kindly provided


by Dr Lawrence Banks and have been described previously (Crook et al., 1991). DNA transfections for transient expression assays were performed by the Lipofectin (Life Technologies, Grant Island, NY, USA) method in HaCaT human immortalized keratinocytes and NIH3T3 ®broblasts, and by Superfect (QIAGEN, Valencia, CA, USA) method in the case of MEF p537/7 and p53+/+ ®broblasts, according to the manufacturer's instructions. The VEGF DNAs (1.5 mg), and the pJ4O16E6 or pJ4O (3 mg) plasmids were co-transfected into target cells grown to 40 ± 50% con¯uence in six-well plates. A plasmid (pRL-CMV) containing the CMV immediate-early enhancer/promoter region coupled to renilla luciferase (5 ng) was also co-transfected as an internal control. After the incubation time recommended by the manufacturer, the cells were washed twice with medium containing serum and cultured for 48 h under standard conditions. The cells were then lysed and assayed for ®re¯y and renilla luciferase activities separately using a DoubleLuciferaseTM Assay System (PROMEGA, Madison, WN, USA) and a Lumat LB 9501 luminometer (EG&G Berthold, Germany). The activity of the VEGF promoter manifested by ®re¯y luciferase activity was determined after normalization for renilla luciferase activity. All transfections were repeated

at least three times and the results are presented as normalized ®re¯y activity+s.e.m.

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Statistics Analysis was performed using the GraphPad Prism Software (GraphPAD, Inc., San Diego, CA, USA). The unpaired Student's t-test was used for all comparisons and in the case of multiple comparisons a one-way ANOVA test was used, coupled to a Newman-Keuls or a Dunnet post test as needed.

Acknowledgments We are grateful to Dr Lawrence Banks for providing J4O16E6 and PJ4O plasmids. We gratefully thank Dr Larry Donehower and Dr Yaacov Ben-David for help in providing MEF p53+/+ and p537/7 cells. Dr Federico DeMarco's help in providing HPK1A cells is also highly appreciated. Ms Lynda Woodcock and Mrs Cassandra Cheng provided excellent secretarial assistance. This work was supported by grants from the Medical Research Council of Canada (MT-5815) and National Institutes of Health, USA (CA-41233) to RS Kerbel.

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