Involvement of RB Gene Family in Tumor Angiogenesis

C Gabellini, D Del Bufalo and G Zupi

Experimental Chemotherapy Laboratory, ‘Regina Elena’ Cancer Institute, Rome, Italy

Angiogenesis, the development of new blood vessels from chronic inflammation, tumor progression and metasta- pre-existing vessels, represents a fundamental step in tization (Folkman, 2003). The induction of vasculature tumor progression and metastatization.The induction is required for the growth of the tumor mass, and to of vasculature is required for growth of the tumor mass, ensure an adequate supply of oxygen and metabolites to to ensure an adequate supply of oxygen and metabolites to the tumor beyond a critical size (Bergers and Benjamin, the tumor beyond a critical size.Tumor angiogenesis is a 2003). Neovascularization is controlled by a balance highly regulated process that is controlled physiologically between several factors promoting or suppressing by the tumor microenvironment and genetically by altera- angiogenesis and the induction of the so-called ‘angio- tion of several oncogenes or tumor suppressor genes.We genic switch’ is determined by proangiogenic factors will focus on recent demonstrations regarding the overcoming the antiangiogenic ones (Folkman, 1995; involvement of the retinoblastoma family proteins (phos- Beck and D’Amore, 1997; Ferrara, 2000; Kerbel, 2000; phorylated retinoblastoma (pRb), p107 and pRb2/p130) Li et al., 2000, Tonini et al., 2003). During the initial at different levels of the angiogenic process.pRb and activation stage, tumor and/or epithelial cells secrete its homologs can regulate the expression of pro- and angiogenic factors such as vascular endothelial growth antiangiogenic factors, such as the vascular endothelial factor (VEGF), basic fibroblast growth factor (bFGF), growth factor, through an E2F-dependent mechanism. platelet-derived growth factor and interleukin-8 (IL-8), Moreover, pRb is able to modulate also the transcriptional which stimulate endothelial cell proliferation, migration activity of several angiogenesis-related factors like HIF-1, and differentiation within the tumor bed (Hanahan, Id2 and Oct-1.pRb2/p130 is required for both differ- 1997; Maisonpierre et al., 1997; Carmeliet and Collen, entiation and mobilization of bone marrow-derived 2000). In later stages, tube formation and vessel matura- endothelial cell precursors and endothelial sprouting from tion lead to vessel remodeling and apoptosis, which neighboring vessels.The involvement of the pRb pathway is regulated by transforming growth factor-b and in the angiogenesis process has also been demonstrated by mesenchymal cells, in the absence of endothelial cell different cellular models expressing viral oncoproteins, proliferation (Pepper et al., 1991; Beck and D’Amore, like human papilloma virus.Moreover, some natural 1997; Taipale and Keski-Oja, 1997). and synthetic compounds demonstrate their antiangiogenetic activity with a mechanism of action involving pRb. Finally, the possible prognostic value of immunohisto- chemical evaluation of pRb and/or pRb2/p130 expression Modulation of angiogenesis involving the retinoblastoma/ can represent a useful tool for the characterization of the E2F pathway in tumor and endothelial cells angiogenic phenotype of specific tumor histotypes. Oncogene (2006) 25, 5326–5332. doi:10.1038/sj.onc.1209631 It has been reported that several oncogenes or tumor suppressor genes have a role in angiogenesis, modulat- Keywords: cancer; angiogenesis; retinoblastoma family ing the expression of pro- and anti-angiogenic factors. proteins Activating mutations in Kras and Hras upregulate VEGF expression and downregulate expression of the angiogenesis inhibitor thrombospondin 1 (TSP-1) (Rak et al., 2000; Udagawa et al., 2002; Watnick et al., 2003). Moreover, wild-type p53 protein normally inhibits Introduction tumor angiogenesis by upregulating TSP-1 (Dameron et al., 1994), inducing degradation of hypoxia-inducible Angiogenesis, the development of new blood vessels factor-1a (HIF-1a) (Ravi et al., 2000), suppressing from pre-existing vessels, represents a fundamental step transcription of VEGF and downregulating bFGF- in several physiological and pathological conditions like binding protein expression (Sherif et al., 2001), whereas embryonic development in utero, wound healing, overexpression of antiapoptotic bcl-2 oncogene increases tumor angiogenesis upregulating VEGF expression (Del Bufalo et al., 1997; Biroccio et al., 2000) Correspondence: Dr G Zupi, Experimental Chemotherapy Labora- tory, ‘Regina Elena’ Cancer Institute, Via delle Messi d’Oro, 156, and enhancing HIF-1 transcriptional activity (Iervolino Rome, 00158 Italy. et al., 2002; Trisciuoglio et al., 2005). In recent years, E-mail: [email protected] there has been experimental evidence demonstrating an Involvement of RB family in angiogenesis C Gabellini et al 5327 involvement of the retinoblastoma (RB) proteins family 2000) and it determines a regression of established in different aspects of the angiogenesis process. The tumor xenografts (Howard et al., 1998). Furthermore, product of the RB susceptibility gene, phosphorylated pRb2/p130 overexpression is able to inhibit angiogen- retinoblastoma (pRb), like the two closely related esis, determining a significant decrease of blood vessel proteins, p107 and pRb2/p130, negatively regulate the density inside tumor grafts. Indeed, pRb2/p130 expres- progression from the G1 to the S phase of the cell cycle. sion reduces VEGF mRNA level and protein expression These proteins are characterized by the presence of a both in vitro and in vivo, downregulating VEGF bipartite pocket structure (A/B domains), which is promoter activity through an indirect mechanism necessary for interaction with E2F transcription factors, (Table 1). A direct transcriptional control of pRb2/ viral oncoproteins and other LXCXE motif-containing p130 has been excluded by the authors because VEGF cellular proteins (Grana et al., 1998; Mulligan and promoter does not contain any responsive site of Jacks, 1998; Classon and Dyson, 2001). All three pocket regulation for pRb2/p130 (Claudio et al., 2001). proteins are substrates of cyclin-dependent kinases pRb2/p130 has also been found to cooperate with (CDKs) and are phosphorylated in a cell cycle- CDK inhibitor p27kip1 to ensure angiogenic homeostasis dependent manner (Grana et al., 1998; Mittnacht, at multiple levels. Mice defective in both pRb2/p130 and 1998): sequential phosphorylation of the pRb protein p27kip1 are not able to form new vessels properly, leading by CDK4/6 and CDK2 leads to dissociation of Rb–E2F to an inability to sustain the growth of tumor xenografts complexes, resulting in the activation of E2F-dependent (Vidal et al., 2005). Nevertheless, p130/pRb2 and p27kip1 transcription and cell cycle progression (Weinberg, are required for endothelial cell differentiation induced 1995; Harbour and Dean, 2000a). In addition to their by VEGF in vitro. Impaired angiogenesis was found role in regulating cell proliferation through the E2F to be associated with defects in the VEGF-induced pathway, pRb family members are able to affect the expansion of myeloid cells and VEGF-induced mobili- chromatin structure and function (Harbour and Dean, zation of both myeloid and endothelial precursors. In 2000b; Nielsen et al., 2001). pRb and its related proteins fact, in addition to endothelial sprouting from neighbor- have overlapping as well as unique cellular functions. ing vessels, the angiogenic switch stimulates bone For example, the RB protein family differs in their marrow-derived precursors to proliferate and induces ability to interact with the various members of the the mobilization of these cells into circulation to reach E2F family. Whereas pRb can interact with E2F1–4, the tumor bed (Lyden et al, 2001; Heissig et al., 2002). both pRb2/p130 and p107 associate with E2F4 and Cyclin E seems to be the fundamental common target E2F5 (Dyson, 1998; Trimarchi and Lees, 2002). In for p130/pRb2 and p27kip1 in the angiogenesis process, general, there is experimental evidence which suggests confirming the involvement of pRb2/p130 in coopera- that pRb2/p130 and p107 are more closely related to tion with p27kip1 in a negative feedback regulatory loop one another than to pRb (Classon and Dyson, 2001). with cyclin E in the modulation of cellular proliferation Through a unique motif in the spacer region, the two (Howard et al., 2000). pRb-related proteins share the ability to bind As described above, hypophosphorylated Rb proteins and directly inhibit the activity of CDK2/cyclin E bind E2F factors and inhibit their transcription activity and CDK2/cyclin A complexes (De Luca et al., 1997; on the promoters of many genes involved in DNA Lacy and Whyte, 1997; Woo et al., 1997; Castano replication and additional cellular functions such as et al., 1998). apoptosis and angiogenesis. Overexpression of E2F1 is Functionally, all RB family members show growth- associated with increased tumor cell invasiveness and suppressive properties. Reconstitution of pRb expres- metastatic progression (Banerjee et al., 2000; Zhang sion in various Rb-defective human cancer cell lines et al., 2000) and E2F1 target genes include several genes determines the suppression of tumorigenicity in nude involved in the angiogenesis process such as bFGF, mice and also correlates with the inhibition of tumor cell fibroblast growth factor receptor 3 (FGFR3), matrix invasion in vitro (Li et al., 1996). Moreover, pRb2/p130 metalloproteinase 16 (MMP16) and VEGF-B (Stanelle expression suppresses tumor growth in vivo by inhibiting et al., 2002), through a direct or indirect transcription tumor formation in nude mice (Claudio et al., 1994, activity on their promoter.

Table 1 Involvement of RB family members in angiogenesis modulators expression RB family member Angiogenesis modulators Mechanism References pRb VEGF Transcriptional level Wegiel et al. (2005) Transcriptional level via Id2 Lasorella et al. (2005) FGFR-1 Transcriptional level via E2Fs 1–3 Tashiro et al. (2003) IL-8 Transcriptional level via Oct-1 Zhang et al. (1999) HRE-responsive genes Transcriptional level via HIF-1 Bardos and Ashcroft (2005) Beischlag et al. (2004) pRb2/p130 VEGF Transcriptional level (indirect) Claudio et al. (2001)

Abbreviations: FGFR-1, ﬁbroblast growth factor receptor 1; HIF-1, hypoxia-inducible factor 1; HRE, hypoxia-responsive elements; Id2, inhibitor of differentiation 2; IL-8, interleukin 8; Oct-1, octamer 1; RB, retinoblastoma; VEGF, vascular endothelial growth factor.

Oncogene Involvement of RB family in angiogenesis C Gabellini et al 5328 Another evidence supports the role of pRb in the The ability of pRb to modulate HIF-1 transcriptional regulation of proangiogenic factor expression at the activity is borne out by studies on the thyroid hormone transcriptional level (Table 1). A functional pRb receptor/RB-interacting protein 230 (TRIP230), a protein is necessary for cyclin A1 to induce the autocrine transcription coactivator that is negatively regulated expression of VEGF in prostate cancer, through by pRb binding (Chang et al., 1997). Under hypoxic an upregulation in VEGF promoter activity (Wegiel conditions, TRIP230 is recruited by HIF-1b/ARNT on et al., 2005). HRE sequences and it is indispensable as a transcription The pRb/E2F-1 pathway may also be indirectly coactivator for HIF-1-regulated genes, in particular the involved in the regulation of angiogenesis through VEGF gene (Beischlag et al., 2004). Altogether, the molecules capable of modulating its activity. For above experimental evidence supports the role of pRb in example, overexpression of cyclin D1, which binds to modulating HIF-1 transcriptional activity, but further and activates the CDK4 and CDK6, renders serum- investigation is needed to comprehend the molecular starved fibroblast responsive to bFGF through a mechanisms involved in such phenomenon. mechanism involving the pRb/E2F-1 pathway. An More recently, it has been demonstrated that in increase of the cyclin D1 level determines an accumu- Rb mutant mice, ectopic expression of the inhibitor of lation of the hyperphosphorylated form of pRb, leading differentiation 2 (Id2) is necessary and sufficient to to pRb inactivation and therefore to the release of induce pituitary tumor angiogenesis through upregula- a transcriptionally active E2F-1 factor, responsible tion of VEGF expression (Lasorella et al., 2005). Id2 for the activation of the fibroblast growth factor belongs to a family of proteins whose function in receptor 1 (FGFR-1) promoter. E2F-2 and E2F-3, the proliferating cells is to inhibit the activity of transcrip- other two members of the E2F family regulated by pRb, tion factors involved in the induction of the differentia- can also increase FGFR-1 promoter activity, whereas tion process. Among Id family members, Id2 specifically E2F-4 and E2F-5, and consequently their negative binds to and inhibits the pRb protein during mouse regulators pRb2/p130 and p107, do not seem to be embryogenesis, in particular in the development of involved in the modulation of FGFR-1 expression the nervous system and hematopoiesis (Iavarone et al., (Tashiro et al., 2003). 1994; Toma et al., 2000). Moreover, Rb-mutated Id2- null pituitary tumors show impaired vascularization and altered VEGF and HIF-1a expression (Lasorella et al., 2005). Id2 could represent one of the links between pRb Modulation of angiogenesis-related transcription and the angiogenesis process: the loss of pRb could factors by pRb result in the activation of Id2 signaling, thus contribut- ing to the angiogenic switch of Rb-null tumors. Several evidences support the role of pRb in regulating Two other members of the Id family, Id1 and Id3, the transcriptional activity of factors involved in angio- share with Id2 the ability to modulate the angiogenic genesis process (Table 1). Recently, the pRb protein process (Lyden et al., 1999). has been identified as a novel interactive partner of HIF- pRb can also interact with another transcription 1a (Budde et al., 2005), a transcription factor induced factor, Octamer 1, inducing an increased secretion of under hypoxic conditions and involved in angiogenesis proangiogenic factor IL-8 through a negative regula- (Semenza, 2001). Cells respond to hypoxia by inducing tion of its DNA-binding activity to the promoter of different protective mechanisms, among them the sta- IL-8 (Zhang et al., 1999). These data contrast with the bilization of the HIF-1a protein. Together with the antiangiogenic role of pRb described above, however, it constitutively expressed HIF-1b/ARNT (aryl hydrocar- should be underlined that the functional significance of bon receptor nuclear translocator), HIF-1a builds an IL-8 increase in pRb-defective tumor cell lines the heterodimer HIF-1, a transcription factor that binds following reconstitution of pRb expression has not been hypoxia-responsive elements (HRE) sequences present investigated by the authors. As a matter of fact, a link in the enhancer or promoter regions of different genes between an increase in the IL-8 level and the possibility involved in glycolysis and angiogenesis. The inter- that pRb reconstitution could modulate the angiogenic action between HIF-1a and pRb exerts functional phenotype of those tumor cell lines has not been consequence for both partners. Hypoxia induces a demonstrated. growth arrest correlated to an accumulation of the hypophosphorylated form of pRb (Ludlow et al., 1993; Amellem et al., 1996; Krtolica and Ludlow, 1996; Danielsen et al., 1998), which is recruited to HRE sites Modulation of angiogenesis by oncoviral proteins and specifically enhances transcriptional activation of targeting retinoblastoma family members HIF-1a (Budde et al., 2005). These data seem to be in contrast with the widely demonstrated antiangiogenic The involvement of the pRb pathway in the angiogen- role of pRb but, on the other hand, the accumulation of esis process is also demonstrated by different cellular HIF-1a induced by hypoxic conditions partially reverses models expressing viral oncoproteins. Adenovirus the repressor function of pRb on E2F-dependent E1A, SV40 large T antigen and human papillomavirus transcription, suggesting a role for HIF-1a in cell cycle (HPV) E6/E7 proteins specifically interact with different progression following reoxygenation. cellular proteins, like RB family members and p53,

Oncogene Involvement of RB family in angiogenesis C Gabellini et al 5329 inhibiting their regulatory function on cell cycle metabolism and detoxification and very recently demon- progression (Munger and Howley, 2002). The infection strated to be involved in the regulation of angiogenesis of primary foreskin keratinocytes with recombinant (Miyashita and Sato, 2005). In serum-starved condi- retrovirus-expressing HPV-16 E6 and E7 oncoproteins tions, all RB family members, together with E2F4 and targeting p53 and pRb, respectively, not only results in E2F5, associate with the hMT1G promoter, leading to immortalization of these cells but also alters the profile its repression. VEGF stimulation leads to a dissociation of expression of different angiogenic factors (Toussaint- of pRb, as well as p130/pRb2 and p107, from hMT1G, Smith et al., 2004). HPV-16 E6 and E7 proteins increase reduces the binding of E2F4 and E2F5 and increases the the secretion of proangiogenic factors, VEGF and IL-8, binding of E2F1, E2F2 and E2F3. These results suggest and downregulate the expression of negative regulators the possibility that VEGF stimulation could affect the of angiogenesis such as TSP-1, maspin, a disintegrin binding of E2Fs and RB family members to other and metalloproteinase with thrombospondin motifs 1 promoters, which regulate the expression of proteins (ADAMTS1), plasminogen activator and fibronectin 1. more directly involved in the modulation of angiogenic Among these genes, TSP-1, maspin and VEGF phenotype. have also been shown to be regulated by p53 (Ravi pRb is also involved in VEGF-induced neurogenesis. et al., 2000; Zou et al., 2000; Harada et al., 2003), whose In fact, in addition to the well-established proangiogenic role in the angiogenic process has been clearly role of VEGF, this molecule appears to have direct described (Bergers and Benjamin, 2003; Fels and neurotrophic and neuroprotective functions (Sondell Koumenis, 2005). However, the HPV-16 E7 but not et al., 1999; Jin et al., 2000; Sondell et al., 2000) and E6 protein strongly enhances the release of angiogenic more recently to act as a neurogenesis factor (Jin et al., factors tumor necrosis factor-a (TNF-a), IL-1b and 2002). VEGF promotes proliferation of cortical neuron IL-6 by activated macrophages (Le Buanec et al., 1999). precursors enhancing nuclear expression of E2F1, E2F2 These findings sustain the hypothesis that E6 and E7 and E2F3, but not E2F4 and E2F5, and increasing the oncoproteins cooperate to stimulate angiogenesis in level of several cyclins involved in cell cycle transition HPV-16-infected cells, modulating different pathways from the G1 to S phase (Zhu et al., 2003). Therefore, involving p53 and pRb. VEGF may induce neurogenesis with a mechanism similar to the one observed during VEGF-stimulated proliferation of endothelial cells, including an increase of cyclin D1 expression, CDK4 kinase activity, pRb Involvement of retinoblastoma family members in phosphorylation and E2F promoter activity (Pedram endothelial cell response to angiogenic stimuli et al., 1998, 2001; Suzuma et al., 2002). Stimulation of endothelial cells by angiogenic cytokines determines the activation of signal transduction pathways also involving RB family members. In response to Modulation of retinoblastoma family members by the proliferation stimulus of different factors like antiangiogenic factors VEGF, pRb is phosphorylated to allow the progression of endothelial cells into the cell cycle. Many genes, The aim of antiangiogenic therapies is to prevent whose expression is regulated by VEGF, contain E2F- the growth of cancer by blocking the formation of new binding sequences in their promoter (Joshi et al., 2005). blood vessels, suppressing proangiogenic signals or In particular, in human aortic endothelial cells, VEGF increasing inhibitory ones (Kerbel and Folkman, 2002). stimulation affects the binding of pRb family members Several studies about antiangiogenic activity of different and E2Fs factors to the promoter of human metallo- cellular factors, as well as natural compounds, confirm thionein 1G (hMT1G), a protein involved in metal the fundamental role of the RB family in modulating the

Table 2 Modulation of pRb by antiangiogenic factors Antiangiogenic factors Effect References

Natural compounds Interferons Dephosphorylation of pRb at Ser780 Raffaella et al. (2004) VEGI Inhibition of pRb hyper-phosphorylation Yu et al. (2001) Endostatin Inhibition of pRb phosphorylation at Ser795 Hanai et al. (2002) CRFR2 Inhibition of pRb hyper-phosphorylation Bale et al. (2002)

Synthetic compounds Raf-1 peptide Disruption of pRb/Raf-1 interaction Dasgupta et al. (2004) Indomethacin Inhibition of pRb hyper-phosphorylation Pai et al. (2000) Capsaicin Inhibition of pRb hyper-phosphorylation Min et al. (2004) AGM-1470 (TNP-470) Inhibition of pRb hyper-phosphorylation Abe et al. (1994) Rosiglitazone Inhibition of pRb hyper-phosphorylation Sheu et al. (2006)

Abbreviations: CRFR2, corticotropin-releasing factor receptor 2; VEGI, vascular endothelial growth inhibitor.

Oncogene Involvement of RB family in angiogenesis C Gabellini et al 5330 angiogenesis process. In particular, pRb is involved in Also, some synthetic compounds demonstrate their the mechanism of action of natural and synthetic antiangiogenic activity with a mechanism of action compounds such as interferon, vascular endothelial involving pRb. Resulting from evidence that Raf-1 can growth inhibitor (VEGI), endostatin and several pep- bind to and inactivate the pRb protein (Wang et al., tides (Table 2). 1998), a peptide corresponding to the Raf-1 amino-acid Interferons, besides their role in viral interference, residue involved in binding to pRb was synthesized. This show a potent antiangiogenic activity downregulating Raf-1 peptide is able to disrupt the physical interaction the expression of several proangiogenic factors such as between Raf-1 and pRb, both in vitro and in vivo, bFGF (Singh et al., 1995), matrix metalloproteinases and demonstrates a potent antiangiogenic activity 2 and 9 (MMP-2 and MMP-9) (Gohji et al., 1994; (Dasgupta et al., 2004). In in vitro angiogenesis system, Kato et al., 1995) and IL-8 (Singh et al., 1996). the Raf-1 peptide inhibits VEGF-induced capillary tube Overexpression of interferon IFI16 inhibits endothelial formation modulating endothelial cell adhesion, inva- cell proliferation, chemotaxis, invasion and tube mor- sion and migration and determining a significant inhi- phogenesis induced by VEGF, through a mechanism bition of VEGF-induced pRb phosphorylation. involving pRb. Indeed, IFI16 exerts its antiangiogenic Moreover, the Raf-1 peptide inhibits tumor growth in activity determining a dephosphorylation of pRb at Ser nude mice, in association with a significant reduction 780, leading to its activation. Moreover, pRb function- of tumor vascularization. These preliminary studies ality is indispensable for IFI16 to exert its antiangio- support the role of pRb in the angiogenic process and genic activity: in human endothelial cells immortalized sustain the possibility that the Raf-1 peptide, or other with HPV-16 E6 and E7 oncogenes IFI16 loses its ability compounds capable of disrupting the interaction be- to inhibit proliferation and tube morphogenesis and tween pRb and Raf-1, could have potential therapeutic enhances some parameters of in vivo angiogenesis value as antineoplastic and antiangiogenic agents. (Raffaella et al., 2004). Indomethacin, a non-steroidal anti-inflammatory pRb is also involved in the mechanism of action of drug with antiangiogenic properties in vitro, is able to VEGI, a member of the tumor necrosis factor family, inhibit basal and bFGF-induced endothelial cell pro- which is primarily expressed in endothelial cells (Zhai liferation through a modulation of the pRb pathway. et al., 1999). VEGI induces growth arrest in endothelial Together with the inhibition of extracellular signal- cells synchronized in the G0/G1 phases of the cell cycle, regulated protein kinase 2 (ERK2) activity and nuclear preventing their progression to the S phase through translocation (Jones et al., 1999), indomethacin stimu- inhibition of pRb hyperphosphorylation by CDKs lates the expression of cyclin kinase inhibitors p21waf1/cip1 (Yu et al., 2001). In the presence of VEGI, the activity and p27kip1, inhibits cyclin D1 and cyclin E expression of CDK4 and CDK6 kinase is inactivated, indicating determining, at least, a reduction of pRb phosphoryla- a possible role of VEGI in the activation of inhibitors tion (Pai et al., 2000). of these CDKs, such as p27kip. Nevertheless, VEGI also In a similar way, the antiproliferative activity of determines a decrease of CDK2 protein level, leading to capsaicin, an alkaloid compound obtained from red the inhibition of its activity. pepper, on endothelial cells is characterized by a down- It has been demonstrated that pRb is involved in the regulation of VEGF-induced cyclin D1 expression mechanism of action of endostatin, the carboxyl- associated with a decrease of pRb hyper phosphoryla- terminal fragment of the XVIII collagen. Endostatin tion. Unlike indomethacin, antiangiogenetic effect of shows a potent antiangiogenic activity (O’Reilly et al., capsaicin is ERK-independent but it is associated with 1997) antagonizing proangiogenic signals of VEGF and an inhibition of the AKT/endothelial nitric oxide bFGF (Karumanchi et al., 2001) and inhibiting synthase signaling pathways (Min et al., 2004). endothelial cell migration and proliferation, through Also another synthetic compound, the fumagillin an induction of cell cycle arrest in the G1 phase analog AGM-1470 (TNP-470), inhibits endothelial cell (Dhanabal et al., 1999). Endostatin downregulates proliferation and tumor-induced vascularization in vivo cyclin D1 expression at the transcriptional level, (Ingber et al., 1990; Farinelle et al., 2000) through a resulting in an inhibition of hyper phosphorylation of mechanism affecting pRb phosphorylation. AGM-1470 the pRb protein induced by VEGF and bFGF (Hanai decreases DNA synthesis and strongly suppresses growth et al., 2002). factor-induced hyperphosphorylation of the pRb protein. Through a similar mechanism, corticotropin-releasing This compound exerts its antiangiogenetic activity also factor receptor 2 (CRFR2), a G-protein-coupled mem- inhibiting growth factor-stimulated activation of cdc2 and brane receptor, which is expressed in both endothelial CDK2 kinases and downregulating the mRNA expression and smooth muscle cells in the vasculature, may of selective members of cyclins/CDKs complexes such as inhibit VEGF- and bFGF-induced angiogenesis and cyclin A, cyclin E and cdc2 without affecting CDK2, play a fundamental role in the physiology of tissue CDK4 and cyclin D1 mRNA level (Abe et al., 1994). vascularization. In fact, the binding of CRFR2 with More recently, rosiglitazone, an insulin sensitizer, has its ligand determines an inhibition of cellular prolifera- been shown to affect VEGF-induced endothelial cell tion, correlated with a dose-dependent inhibition of proliferation, migration and morphogenesis altering pRb phosphorylation at the Ser 795 residue, and an several proteins involved in cell cycle control and inhibition of VEGF-stimulated formation of capillary- determining a decrease of pRb phosphorylation (Sheu like structures in vitro (Bale et al., 2002). et al., 2006).

Oncogene Involvement of RB family in angiogenesis C Gabellini et al 5331 Correlation of retinoblastoma family members and VEGF (Ludovini et al., 2004). Furthermore, in both hepato- expression in human cancer cellular carcinoma and endometrial cancer specimens, pRb2/p130 expression is inversely correlated with Human RB is a solid well-vascularized tumor (Madigan VEGF level and tumor aggressiveness (Claudio et al., and Penfold, 1997). The first studies about RB- 2004; Sanseverino et al., 2006). These evidences associated angiogenesis were developed using aqueous suggest that the correlation between pRb, or pRb2/ humors from enucleated RB-bearing eyes, testing their p130, and VEGF expression most probably depends antiangiogenic activity in vitro. This first experimental on the specific cellular context and genetic background approach on human specimens demonstrated that of tumor. aqueous humors from RB patients showed a significant These data suggest the possible use of immunohisto- increase in angiogenic activity compared to control chemical evaluation of pRb and/or pRb2/p130 expres- humors (Albert et al., 1984). Moreover, VEGF mRNA sion as a useful tool for the characterization of the was found to be highly expressed in RB histo- angiogenic phenotype of specific tumor histotypes, logical sections (Kvanta et al., 1996). Also for other providing new prognostic disease-specific information tumor histotypes, evidence of correlation between RB together with conventional pathology. family members expression and angiogenetic markers are emerging. In preinvasive breast pathologies, Acknowledgements pRb expression does not correlate with vascularity (Heffelfinger et al., 2000), whereas immunohisto- We thank Dr Adele Petricca for secretarial assistance in the chemical evaluation of advanced non-small cell preparation of the manuscript and Paula Franke for revising lung cancer biopsy specimens demonstrates a negative the English language. Gabellini C is a recipient of a fellowship correlation between pRb and VEGF expression from Italian Foundation for Cancer Research.

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