Enhanced Expression of Vascular Endothelial Growth Factor (VEGF) Plays a Critical Role in the Tumor Progression Potential Induced by Simian Virus 40 Large T Antigen

Enhanced expression of vascular endothelial growth factor (VEGF) plays a critical role in the tumor progression potential induced by simian virus 40 large T antigen

Alfonso Catalano1, Mario Romano*,2, Stefano Martinotti3 and Antonio Procopio*,1

1Institute of Experimental Pathology, University of Ancona, Faculty of Medicine, Via Ranieri, 60131 Ancona, Italy; 2Department of Human Pathology, University of Messina, 98125 Messina, Italy; 3Department of Oncology and Neuroscience, `G. D'Annunzio' University, Chieti, Italy

Vascular endothelial growth factor (VEGF), an impor- disorders, including mesotheliomas (Kumar-Singh et tant angiogenic factor, regulates cell proliferation, al., 1999). Among these, VEGF, whose crucial role in dierentiation, and apoptosis through activation of its tumor angiogenesis is well established (Hanahan and tyrosine-kinase receptors, such as Flt-1 and Flk-1/Kdr. Folkman., 1996), acts also as a potent mitogen and Human malignant mesothelioma cells (HMC), which survival factor for human malignant mesothelioma have wild-type p53, express VEGF and exhibit cell cells (HMC). In fact, VEGF regulates HMC prolifera- growth increased by VEGF. Here, we demonstrate that tion through its tyrosine-kinase receptors activation early transforming proteins of simian virus (SV) 40, large (i.e. Flt-1 and KDR/¯k-1) (Strizzi et al., 2001). In tumor antigen (Tag) and small tumor antigen (tag), addition, VEGF is the main eector of 5-lipoxygenase which have been associated with mesotheliomas, en- action on HMC survival (Romano et al., 2001). Thus, hanced HMC proliferation by inducing VEGF expres- VEGF plays a key role by regulating multiple sion. SV40-Tag expression potently increased VEGF mechanisms of tumor progression, such as formation protein and mRNA levels in several HMC lines. This of new blood vessels and proliferation of neoplastic eect was suppressed by the protein synthesis inhibitor, cells. cycloheximide. Inactivation of the VEGF signal trans- The development of mesothelioma is associated with duction pathway by expression of soluble form of Flt-1 a history of asbestos exposure (Browne, 2001). inhibited Flk-1/Kdr activation and HMC proliferation However, other factors may intervene in the pathogen- induced by SV40 early genes. Experiments with SV40 esis of this tumor. The presence of nucleic acids and mutants revealed that SV40-Tag, but not -tag, is involved proteins of Simian Virus 40 (SV40) has been observed in the VEGF promoter activation. However, concomitant in most of mesotheliomas (Carbone et al., 1994). expression of SV40-tag enhanced Tag function. In Moreover, SV40 infection represents a negative prog- addition, SV40-Tag expression sustained VEGF induc- nostic cofactor for patients aected by mesothelioma tion in colon carcinoma cell line (CCL)-233, which have (Procopio et al., 2000). SV40 encodes two transforming wild-type p53, but not in CCL-238, which lack functional proteins, the large tumor antigen (Tag) and the small p53. These data indicate that VEGF regulation by SV40 tumor antigen (tag). SV40-Tag, a 90 kDa nuclear transforming proteins can represent a key event in SV40 phosphopolypeptide, is essential for virus growth and signaling relevant for tumor progression. sucient to induce mesothelial cell transformation in Oncogene (2002) 21, 2896 ± 2900. DOI: 10.1038/sj/ the absence of cell lysis (Bocchetta et al., 2000). onc/1205382 Although SV40-Tag displays pleotropic actions on multiple potential mechanisms of cell transformation Keywords: SV40; Tag; VEGF; mesothelioma (DeCaprio, 1999), it has been proposed that it may facilitate cell transformation by binding and inactivat- ing p53 and retinoblastoma (Rb) tumor suppressor proteins (Carbone et al., 1997; De Luca et al., 1997). However, the molecular consequences of these associa- Increased expression of angiogenic cytokines, such as tions are only beginning to be described. ®broblast growth factor (FGF), platelet-derived growth In the present report, we asked whether a relation- factor (PDGF), and vascular endothelial growth factor ship might exist between SV40-related proteins and (VEGF), has been shown in numerous malignant VEGF expression. To this end, SV40-negative HMC (MPP89) (Orengo et al., 1999) were transiently transfected with an SV40-Tag expression vector, pw2dl (Bocchetta et al., 2000). As shown in Figure 1a, a time- *Correspondence: A Procopio; E-mail: [email protected] dependent expression of SV40-Tag was observed. The and M Romano; E-mail: [email protected] Received 26 September 2001; revised 29 January 2002; accepted 7 88 and 90 kDa polypeptides detected by the Tag February 2002 antibody correspond to the previously described Tag SV40 large T antigen regulates VEGF expression A Catalano et al 2897 phosphoisoforms (Paley, 1996). When it was examined lines (results not shown). Notably, VEGF mRNA the eect of SV40-Tag expression on VEGF secretion expression induced by SV40-Tag was suppressed by the in MPP89, a selective temporal relationship between protein synthesis inhibitor cycloheximide (Figure 1d), SV40-Tag expression and increased VEGF production indicating that the accumulation of VEGF mRNA was observed (Figure 1b). Moreover, 96 h after SV40- induced by SV40-Tag is dependent on newly synthe- Tag transfection, MPP89 displayed maximum increase sized proteins. in all VEGF protein isoforms (Figure 1a) and returned In the speci®c case of HMC, VEGF also acts as a to basal levels within 6 days (results not shown). In potent autocrine growth factor. Thus, to establish the contrast, SV40-Tag did not alter the levels of other relevance of VEGF regulation by SV40-Tag, we HMC-derived angiogenic factors, such as FGF-type 2 antagonized VEGF activity in Tag-expressing HMC and PDGF (Figure 1a). SV40-Tag also increased using an adenoviral vector encoding a soluble form of VEGF mRNA levels by ®vefold and eightfold after Flt-1 (Ad.sFlt-1) (Takayama et al., 2000). sFlt-1 48 and 72 h, respectively (Figure 1c). Similar results expression signi®cantly inhibited VEGF accumulation were obtained using three additional distinct HMC in conditioned media of SV40-Tag-expressing MPP89

Figure 1 SV40-Tag expression increased VEGF production in HMC. MPP89 were transfected with 3.0 mg pw2dl. (a) Cellular extracts were collected for the indicated times after transfection. Seventy-®ve mg of soluble protein extracts were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). VEGF, PDGF, and FGF-2 proteins were detected by autoradiography with a polyclonal anti-VEGF antibody (1 : 1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), a polyclonal anti-PDGF antibody (1 : 40 000 dilution; Jerey Weidner, Merck Research Laboratories), or a polyclonal anti-FGF-2 antibody (5 ng/ml; Upstate Biotechnology, Lake Placid, NY, USA), as previously described (Gaetano et al., 2001). For analysis of SV40-Tag expression, 50 mg of protein extract was separated by 7% SDS polyacrylamide gel electrophoresis and Tag proteins were detected with a rabbit polyclonal anti-Tag antibody (Santa Cruz Biotechnology), as described (Carbone et al., 1997). Protein concentration was determined by standard Bradford protein assay (Bio- Rad Lab., Hercules, CA, USA). Extracts from the Tag-expressing 1166 cells and human umbilical vein endothelial cells were used as positive controls (+) for Tag and VEGF expression, respectively. (b) Conditioned media of MPP89 transfected with pw2dl (+ Tag) or with the control plasmid, pw101 (7 Tag) were collected at the indicated times and VEGF was measured using an ELISA assay (R&D System Europe Ltd., Abingdon, UK) according to manufacturer's instructions. Results are from two independent experiments with duplicates. (c) Total RNA was extracted using the TRIzol reagent (Life Technologies, Milan, Italy). RNA (15 ± 20 mg) was resolved using a 1.2% agarose gel containing 6.3% formaldehyde, transferred to a HybondTM-N nylon membrane (Amersham) and hybridized with a 32P-labeled cDNA probe containing the 522 bp of the human VEGF sequence common to all known VEGF isoforms (Oncogene Research Products, Cambridge, MA, USA). All Northern blots were reprobed with random primed 32P-labeled human b-actin cDNA. Fold increase was calculated by densitometry after normalization for b-actin expression. Basal expression at time `0' was assumed as 1.0. RNA from a melanoma cell line (SK-Mel-147) was utilized as a positive control (+). (d) MPP89 transfected with pw2dl for 48 h, were treated with 25 mg/ml of cycloheximide (CHX) for additional 3 h, prior to total RNA extraction. VEGF mRNA was assessed by Northern blot. Results are from one representative experiment (n=2)

Oncogene SV40 large T antigen regulates VEGF expression A Catalano et al 2898 (P=0.03) (Figure 2a). Moreover, it abrogated both Flk-1/Kdr phosphorylation and DNA synthesis induced by SV40-Tag in HMC (Figure 2b,c). These results strongly indicate that VEGF signaling induced by SV40-Tag contributes to cell cycle modulation promoted by SV40. To investigate the mechanisms of VEGF expression by SV40 early genes, a VEGF promoter-luciferase construct, covering the promoter region 72018 to +50 (Finkenzeller et al., 1997), was transiently co-transfected into MPP89 with SV40 plasmids harboring various combinations of the SV40 transforming proteins. As shown in Figure 3a, a signi®cant increase in luciferase activity was denoted following transfection of the plasmid expressing both SV40 oncoproteins (T/t) and the plasmid expressing Tag alone (T/7)(P50.001

Figure 2 Impact of Ad.sFlt-1 on VEGF signaling triggered by SV40-Tag in HMC. Adenovirus vectors were ampli®ed in 293 cells and titered by plaque forming assay according to standard protocols. Viral stocks were maintained at 7808C for long-term storage before use. HMC were transfected with 4.0 mg of pw2dl (+ Tag) or with pw101 (7 Tag), incubated for 24 h, then infected with Ad.sFlt-1 or Ad.Null (50 p.f.u.6cell) for additional 48 h. (a) VEGF levels in conditioned media were measured by Figure 3 Activation of a VEGF promoter construct by SV40 ELISA. Results are from n=3 (*P50.001). (b) Cell lysates were early genes. A VEGF promoter construct (2.0 mg) and plasmids separated by 7.5% polyacrylamide gel electrophoresis and (from 1.0 to 4.0 mg) harboring SV40 early genes: pw2, expressing transferred to nitrocellulose membrane. Blots were exposed to both Tag and tag (T/t); pw2dl, expressing Tag alone (T/7); pw2t, PY20 anti-phosphotyrosine monoclonal antibody (1 : 1000) (Santa expressing tag alone (7/t); and pw101, expressing only a mutated Cruz Biotechnology) for 1 h at room temperature (upper panels), tag (7/7) were co-transfected using the lipofectamine-plus stripped and re-blotted with an anti-¯k-1/Kdr antibody (lower reagent (Life Technologies, Milan, Italy) into MPP89 (a)or panels). The bands were visualized using the ECL Western A549 (ATCC, Manassas, VA, USA) (b). pCDNA3, containing blotting system (Amersham Int., Bucks, UK). Results are from a the CMV immediate-early enhancer/promoter region coupled to single experiment representative of three. (c)46104 cells/ml were b-galattosidase (0.5 mg), was also transfected as an internal seeded into 24-well plates and grown to 70% con¯uency over control. After 24 h from transfection cells were lysed and tested 24 h. Cells were made quiescent with FCS-free medium for 12 h for luciferase activity using the Double-LuciferaseTM Assay before transfection experiments. At the end of the incubations, System (Promega, Milan, Italy). Results were normalized for b- [3H]-Thymidine (0.5 mCi/ml) was added for additional 4 h. Details galattosidase activity, measured by Galacto-Light PlusTM (TRO- of the [3H]-Thymidine uptake assay are reported elsewhere (Strizzi PIX Inc., Bedford, MA, USA) according to manufacturer's et al., 2001). Results are mean+s.d. from n=3 with duplicates instructions. Results represent mean+s.d. from three independent (*P50.05) experiments (*P50.05; **P50.01)

Oncogene SV40 large T antigen regulates VEGF expression A Catalano et al 2899

Figure 4 Analysis of p53 impact on SV40-Tag-induced VEGF expression. (a) MPP89, transfected with the VEGF promoter construct (2.0 mg) together with 3.0 mg of the SV40-Tag-expressing plasmid (+ Tag) or the control plasmid (7 Tag) were exposed to 10 mM of menadione for 8 h. Luciferase activity was determined as described in the legend to Figure 3. Results represent mean+s.d. from n=3 (*P50.05). (b) VEGF release was assessed by ELISA (n=3; *P=0.03). (c) CCL-238 (black bars) and CCL- 233 (gray bars) were transfected as above and VEGF promoter activity determined. Results were from n=3 and P=0.03, respectively). Thus, tag may act sinergis- impact of menadione, a drug that selectively disrupts tically with Tag to activate the VEGF promoter. In Tag/p53 nuclear complex (Gonin et al., 1999), on contrast, the vectors expressing tag alone (7/t) or a VEGF promoter activation induced by SV40-Tag. mutated tag, that did not bind protein phosphatase 2A Menadione drastically reduced either VEGF promoter (7/7), did not in¯uence VEGF transcription. These activity and protein levels induced by SV40-Tag ®ndings are consistent with previous results showing (Figure 4a,b). As expected, menadione increased p53- that tag may enhance cell transformation potential of mediated p21WAF1/CIP1 expression (results not shown). Tag by inhibiting protein phosphatase 2A activity Then, to con®rm that p53 was involved in the SV40- (Bocchetta et al., 2000). Similar eects were observed Tag-mediated VEGF expression, we used a wild-type when the lung cancer-derived cell line, A549, was p53 (+/+) (CCL-233) and a p53-null (7/7) (CCL- transfected with the SV40 plasmids (Figure 3b), 238) colon cancer cell lines, as recipient cells. We indicating that VEGF regulation by SV40 is not observed that SV40-Tag expression sustained VEGF restricted to HMC. Thus, SV40 by its early genes induction in CCL-233, but not in CCL-238 (Figure 4c). expression can stimulate VEGF transcription, but, Together these results indicate that VEGF expression considering that VEGF mRNA accumulation de- induced by SV40-Tag is involved in the cell cycle pended on newly synthesized proteins (see Figure 1d), perturbation caused by p53 inactivation in response to additional factors may intervene. SV40 infection. Binding and inactivation of the tumor suppressor The mechanisms of VEGF regulation remain to be p53 by SV40-Tag has been observed in HMC (Carbone fully investigated. Previous studies have shown that et al., 1997). Moreover, p53 can suppress VEGF p53 represses VEGF transcription by preventing the expression (Zhang et al., 2000). Therefore, we asked binding of Sp-1 to the VEGF promoter (Zhang et al., whether p53 was partially involved in VEGF regulation 2000). More recently the involvement of Src kinase by SV40. To answer this question we ®rst examined the activity in p53 inhibition of VEGF transcription has

Oncogene SV40 large T antigen regulates VEGF expression A Catalano et al 2900 been assessed (Pal et al., 2001). In addition, also p16 In conclusion, we present here the ®rst evidence of a and Rb family members can inhibit VEGF expression molecular link between SV40 expression and VEGF (Claudio et al., 2001). Therefore, it is possible that cell regulation involving p53 inactivation. These ®ndings cycle regulatory proteins generally acting in the G1 may be particularly relevant for a better understanding phase of the cell cycle could have similar eects, or that of the mechanisms of tumor progression and open new these proteins control distinct pathways with a prognostic and therapeutic perspectives for SV40- common end point. Since SV40 simultaneously can related neoplasms such as mesotheliomas. repress both p53 and Rb function to accomplish cellular transformation, VEGF up-regulation could represent a common molecular strategy for SV40 to regulate proliferation and tumor progression. However, the roles of these DNA tumor viruses in tumorigenesis Acknowledgments need to be studied more closely. This work was presented in part at the International Our results suggest that the impact of SV40-Tag on Conference on Malignant Mesothelioma-Therapeutic Op- VEGF expression is abrogated by blocking protein tions and role of SV40: An Update, Chicago, Illinois synthesis and is dependent by p53 status. Thus, the (USA), April, 2001. It was supported by grants from the SV40-Tag/p53 binding may deregulate the synthesis of Associazione Italiana per la Ricerca sul Cancro (AIRC) inhibitors or activators of VEGF transcription. Recent and from the Italian Ministero dell' UniversitaÁ edella observation from our group points at nitric oxide Ricerca Scienti®ca (ex 60%) to A Procopio. A Catalano was supported by a fellowship from FIRC. We thank Dr G synthase 2 as a key regulator of p53-VEGF interac- Finkenzeller and Dr J Abraham for making the VEGF tions in HMC (Catalano et al., unpublished). However, promoter construct, Dr MC Capogrossi for providing the the possibility that SV40-Tag can also induce a subset adenovirus vectors, Dr G Vianale and Dr Strizzi for of transcription factors acting on the VEGF promoter technical assistance and Dr M Bocchetta for providing the in a p53-independent fashion cannot be ruled out. SV40 plasmids.

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