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 Catalano 1 , Mario Romano* ,2 , Stefano Martinotti 3 and Antonio Procopio* ,1 1 Institute of Experimental Pathology, University of Ancona, Faculty of Medicine, Via Ranieri, 60131 Ancona, Italy; 2 Department of Human Pathology, University of Messina, 98125 Messina, Italy; 3 Department of Oncology and Neuroscience, `G. D'Annunzio' University, Chieti, Italy Vascular endothelial growth factor (VEGF), an impor- tant angiogenic factor, regulates cell proliferation, dierentiation, and apoptosis through activation of its tyrosine-kinase receptors, such as Flt-1 and Flk-1/Kdr. Human malignant mesothelioma cells (HMC), which have wild-type p53, express VEGF and exhibit cell growth increased by VEGF. Here, we demonstrate that early transforming proteins of simian virus (SV) 40, large tumor antigen (Tag) and small tumor antigen (tag), which have been associated with mesotheliomas, en- hanced HMC proliferation by inducing VEGF expres- sion. SV40-Tag expression potently increased VEGF protein and mRNA levels in several HMC lines. This eect was suppressed by the protein synthesis inhibitor, cycloheximide. Inactivation of the VEGF signal trans- duction pathway by expression of soluble form of Flt-1 inhibited Flk-1/Kdr activation and HMC proliferation induced by SV40 early genes. Experiments with SV40 mutants revealed that SV40-Tag, but not -tag, is involved in the VEGF promoter activation. However, concomitant expression of SV40-tag enhanced Tag function. In addition, SV40-Tag expression sustained VEGF induc- tion in colon carcinoma cell line (CCL)-233, which have wild-type p53, but not in CCL-238, which lack functional p53. These data indicate that VEGF regulation by SV40 transforming proteins can represent a key event in SV40 signaling relevant for tumor progression. Oncogene (2002) 21, 2896 ± 2900. DOI: 10.1038/sj/ onc/1205382 Keywords: SV40; Tag; VEGF; mesothelioma Increased expression of angiogenic cytokines, such as ®broblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), has been shown in numerous malignant disorders, including mesotheliomas (Kumar-Singh et al., 1999). Among these, VEGF, whose crucial role in tumor angiogenesis is well established (Hanahan and Folkman., 1996), acts also as a potent mitogen and survival factor for human malignant mesothelioma cells (HMC). In fact, VEGF regulates HMC prolifera- tion through its tyrosine-kinase receptors activation (i.e. Flt-1 and KDR/¯k-1) (Strizzi et al., 2001). In addition, VEGF is the main eector of 5-lipoxygenase action on HMC survival (Romano et al., 2001). Thus, VEGF plays a key role by regulating multiple mechanisms of tumor progression, such as formation of new blood vessels and proliferation of neoplastic cells. The development of mesothelioma is associated with a history of asbestos exposure (Browne, 2001). However, other factors may intervene in the pathogen- esis of this tumor. The presence of nucleic acids and proteins of Simian Virus 40 (SV40) has been observed in most of mesotheliomas (Carbone et al., 1994). Moreover, SV40 infection represents a negative prog- nostic cofactor for patients aected by mesothelioma (Procopio et al., 2000). SV40 encodes two transforming proteins, the large tumor antigen (Tag) and the small tumor antigen (tag). SV40-Tag, a 90 kDa nuclear phosphopolypeptide, is essential for virus growth and sucient to induce mesothelial cell transformation in the absence of cell lysis (Bocchetta et al., 2000). Although SV40-Tag displays pleotropic actions on multiple potential mechanisms of cell transformation (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- tions are only beginning to be described. In the present report, we asked whether a relation- ship might exist between SV40-related proteins and 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- dependent expression of SV40-Tag was observed. The 88 and 90 kDa polypeptides detected by the Tag antibody correspond to the previously described Tag Oncogene (2002) 21, 2896 ± 2900 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc *Correspondence: A Procopio; E-mail: procopio@popcsi.unian.it and M Romano; E-mail: mromano@unich.it Received 26 September 2001; revised 29 January 2002; accepted 7 February 2002