Biochimica et Biophysica Acta 1654 (2004) 23 37 www.bba-direct.

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Review

Stimulation of angiogenesis by Ras proteins

Onno Kranenburg a,*, Martijn F.B.G. Gebbink b, Emile E. Voest b
b

Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Department of Medical Oncology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Received 7 April 2003; accepted 3 September 2003

Abstract Cells that have acquired a proliferative advantage form islets of hyperplasia during the initial stages of tumor development. Like normal cells, they require oxygen and nutrients to survive and proliferate. The centre of the islets is characterized by low oxygen pressure and low pH, conditions that stimulate the sprouting of new capillaries from nearby vascular beds. It is now well established that neovascularisation (angiogenesis) of the hyperplasias is essential for further development of the tumor. The family of ras oncogenes promotes the initiation of tumor growth by stimulating tumor cell proliferation, but also ensures tumor progression by stimulating tumor-associated angiogenesis. Oncogenic Ras proteins stimulate a number of effector pathways that culminate in the transcriptional activation of genes that control angiogenesis. Moreover, Ras signaling leads to stabilization of the produced mRNAs and, possibly, to enhanced initiation of their translation. In this review we describe the mechanisms that underlie Ras regulation of vascular endothelial growth factor (VEGF), cyclooxygenases (COX-1/-2), thrombospondins (TSP-1/-2), urokinase plasminogen activator (uPA) and matrix metalloproteases-2 and -9 (MMP-2/-9). As a result of these Ras-regulated changes in gene expression, the tumor cells cause stimulation of endothelial cells in nearby vascular beds (directly via VEGF, and indirectly via COX-produced prostaglandins) and promote remodeling of the extracellular matrix (by lowering TSP and increasing uPA/MMPs). The latter effect makes growth factors available for endothelial cell activation and migration. In addition, tumor cell-activated stromal cells also contribute to the stimulation of angiogenesis by further enhancing the production and secretion of proangiogenic factors into the tumor stroma. D 2004 Elsevier B.V. All rights reserved.
Keywords: Ras; Angiogenesis; VEGF; Cyclooxygenase; Thrombospondin; Metalloprotease; Urokinase

1. Introduction A series of genetic alterations that turn proto-oncogenes into oncogenes and active tumor suppressor genes into inactive ones underlies the complex process of tumor development. Early studies focused on identifying the transforming genes in the genomic DNA of human tumor cells. Some of these transforming oncogenes were found to be the human homologues of oncogenes in the Harvey and Kirsten Rat sarcoma viruses (v-H-ras, v-K-ras) [1 3]. A

Abbreviations: TSP, thrombospondin; COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; VEGF, vascular endothelial growth factor; MMP, matrix metalloprotease; uPA, urokinase-type plasminogen activator; FTI, farnesyltransferase inhibitor * Corresponding author. Department of Medical Oncology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Tel.: +31-30-2506681; fax: +31-30-2523741. E-mail address: o.kranenburg@azu.nl (O. Kranenburg). 0304-419X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2003.09.004

related transforming gene was subsequently isolated from a human neuroblastoma cell line (SK-N-SH) and was designated N-ras [4,5]. Activating mutations in all three ras genes, c-H-ras, c-K-ras2 (c-K-ras1 is a pseudogene) and c-N-ras, are found in a large variety of human tumors [6]. Distinct types of tumors are associated with mutations in distinct Ras isoforms. K-ras2 mutations are frequently found in carcinomas of the colon, the lung (non-small cell lung carcinoma) and the pancreas, whereas N-ras mutations are primarily found in melanomas and hematologic cancers and H-ras mutations are uncommon in human tumors [6]. The reason(s) for these differences are still largely unclear. Wild-type Ras proteins control signal transduction pathways downstream from transiently activated (ligand-bound) tyrosine kinase receptors and G protein-coupled receptors [7,8]. Ras activation occurs through the stimulated exchange of bound GDP for GTP. GTP-bound Ras activates

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several signal transduction cascades that lead to changes in gene expression and cell motility. Thereby, Ras signaling controls fundamental cell biological processes like proliferation, differentiation, survival and invasion. Mutations in the ras genes that are found in tumors (commonly in codons 12, 13 or 61) greatly impair the ability of Ras to hydrolyze bound GTP and thereby its intrinsic ability to switch off. As a result, mutant (GTPase-deficient) Ras proteins constitutively stimulate signal transduction through their effector pathways. Mutational activation of ras genes has been detected in pre-malignant hyperplasias in the colon, thyroid, lung and bone marrow [6,9 13]. Thus, Ras protein activation occurs early during tumor development in man. Several observations suggest that mutational activation of one of the ras genes is, in fact, an initiating event in tumor development. First, many of the tumors induced by mutagenic compounds in mice harbor activating mutations in one of the three ras genes and these mutations can be detected at the very early stages of tumor development [14 20]. Second, studies in mice that are genetically modified to (conditionally) express K-ras2 or H-ras oncogenes have further and unequivocally established that activated Ras promotes the initiation and progression of several types of tumors [21 26]. Moreover, the expression of activated H-Ras is also required for the maintenance of experimental (melanoma) tumor growth [27]. Constitutive Ras signaling promotes tumor cell proliferation, survival and invasiveness but also causes modulation of the behavior of non-tumor cells in the microenvironment. Of particular relevance to this review is the capacity of mutant Ras-containing tumor cells to stimulate endothelial cells in nearby capillary beds to grow out and form new blood vessels (angiogenesis) [28]. Growing tumors depend on angiogenesis for sufficient supply of oxygen and nutrients [29]. This is true for most solid tumors, but also for hematologic cancers that grow in the bone marrow [30]. The first observation that activated Ras stimulates tumorassociated angiogenesis came from a study in which the contribution of different oncogenes to tumorigenesis was studied in vivo, in a reconstituted mouse prostate gland infected with distinct oncogene-expressing retroviruses [31]. The authors found that activated Ras not only caused local hyperplasia but also hypervascularisation of the gland with more than a 10-fold increase in cytologically normal blood vessels. Interestingly, both the mutational activation of Ras and the stimulation of angiogenesis are early pre-malignant events when examined in several clinical and experimental settings [14,21,31 37]. In this review, the mechanisms underlying Ras-stimulated angiogenesis will be discussed. In particular, we have attempted to clarify the causal relationship between Rasstimulated signal transduction pathways and the angiogenesis-related phenomena that are observed in tumors and cell lines expressing the distinct activated Ras isoforms.

2. Vascular endothelial growth factor (VEGF) in Rasstimulated angiogenesis 2.1. Oncogenic Ras promotes VEGF expression VEGF-A is one of the most potent angiogenesis-stimulating growth factors, and has become a key target in antitumor therapy [38 40]. Tumors harboring mutant ras often express high levels of VEGF. Retrospective studies have shown a correlation between the presence of oncogenic Kras2 and high VEGF levels in patients with pancreas carcinoma and non-small cell lung carcinoma [41,42]. Furthermore, elevated VEGF levels and increased bone marrow vascularisation are associated with poor prognosis in hematologic malignancies [30,43 45], although in these cases the correlation with N-ras gene mutations (frequently observed in these cancers) has not yet been evaluated. Tumor-derived cell lines expressing activated K-Ras as well as cell lines transfected with an activated H-ras gene show increased synthesis and production of VEGF-A [46 48], but not VEGF-B or VEGF-C [49,50]. Conversely, inhibition of mutant K-ras2 gene expression in human colon cancer cells, either through disruption of the gene itself (by homologous recombination), through expression of a hammerhead ribozyme (specifically cleaving mutant K-Ras mRNA) or through antisense oligonucleotides, reversed the up-regulation of VEGF expression [48,51,52]. Thus, activated K-Ras is instrumental in elevating VEGF expression in colon cancer cells. What is the mechanism of Ras-stimulated VEGF production? The best-known VEGF-regulating transcription factor is hypoxia-inducible factor 1 (HIF-1), which is involved in the activation of VEGF synthesis under hypoxic conditions [53]. HIF-1 a is normally bound by the Von Hippel Lindau (VHL) tumor suppressor protein and is thereby targeted for degradation. HIF-1 a hydroxylation, as it occurs under normoxic conditions, is required for the interaction with VHL. Hypoxia leads to diminished HIF-1 a hydroxylation, release of HIF-1 a from VHL, and to expression of HIF-1 target genes, including VEGF [54 56]. Thus, HIF-1-dependent transcription is activated under hypoxic conditions. Interestingly, there is evidence to suggest that Ras activation modulates this oxygen-sensing signal transduction cascade. Two Ras-activated signal transduction pathways have been implicated in the control of VEGF expression. The classical Ras>Raf >MEK>ERK1/2 kinase cascade and the Ras >PI(3)K>PDK>PKB pathway independently control VEGF transcription in cells expressing oncogenic Ras. HIF-1 a is directly phosphorylated by ERK1/2, resulting in enhanced transactivation potential and stimulation of the VEGF promoter [50,57 60]. In addition, Ras signaling is also instrumental in hypoxia-induced stabilization of HIF1 a protein [61], and, in line with this, elevated levels of HIF-1 a are found in fibroblasts and breast cancer cells expressing activated Ras, even under normoxic conditions [62,63]. The mechanism underlying

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this phenomenon is presently unknown but may involve PI(3)-kinase-regulated modulation of HIF degradation [59,62 64]. By regulating HIF-1 protein stability and activity, oncogenic Ras can stimulate VEGF gene transcription through the HIF-binding region in the VEGF promoter. However, this is not the only mechanism. Several other transcription factors on the VEGF promoter are affected by Ras signaling. A critical element in the promoter is a GC-rich stretch ( 88/ 50) that contains consensus binding sites for Sp1, Sp3 and AP2 [60,65]. Stimulation of the classical Ras> Raf >MEK>ERK1/2 pathway, either by expression of oncogenic H-Ras, inducible Raf, or constitutively active MEK, results in enhanced VEGF promoter activity that requires the Sp1/AP2 binding region. Indeed, binding of Sp1 and AP2 to this promoter element is greatly enhanced following expression of Raf [50]. This effect is presumably caused by direct phosphorylation of Sp1 by ERK1/2, as this greatly increases Sp1 binding to DNA [66,67]. VEGF mRNA levels are not solely determined by changes in gene transcription, but also by modulation of mRNA stabilization, presumably via the binding of cellular proteins to the 3VUTR [68,69]. Activation of stress-activated protein kinases (like Jun-kinase (JNK)) may modulate binding of these factors to the VEGF mRNA [69]. Oncogenic Ras stimulates these kinases through the Ras >Rac > MEKK1>JNKK>JNK pathway. VEGF mRNA is indeed three- to fivefold more stable in human tumor cells harboring activated H-Ras (EJ bladder carcinoma) and N-Ras (HT 1080 fibrosarcoma) as well as in murine fibroblasts expressing oncogenic H-ras [70], but it has not been evaluated whether Ras-mediated activation of JNK is responsible for this effect. Tumorigenicity is not only associated with changes in gene expression, but also with a general increase in mRNA translation [71]. The translation initiation factor eIF-4E is under negative control of the 4E-binding protein 1 (4E-BP1). Phosphorylation of 4E-BP1 results in decreased eIF-4E binding and enhanced translation initiation [72]. Interestingly, the kinase cascades that control 4EBP1 phosphorylation, PI(3)K>PDK>PKB>FRAP and Raf >MEK>ERK1/2, are stimulated by Ras [73,74]. Stimulated eIF-4E-dependent protein translation is essential for tumor growth of Rastransformed fibroblasts through inhibition of tumor cell apoptosis [75]. Furthermore, increased VEGF levels in head and neck, breast and bladder cancer correlate with high levels of eIF-4E expression [76 80], and VEGF protein is highly up-regulated in eIF-4E-overexpressing cells [81]. Further work is still required to assess whether the initiation of VEGF mRNA translation is indeed enhanced in tumor cells harboring oncogenic Ras and, if so, whether this is caused by increased 4E-BP phosphorylation. Taken together, Ras regulation of VEGF synthesis is very complex and occurs at multiple levels of transcription and translation (Fig. 1). Furthermore, many different cell lines have been used to study the distinct phenomena outlined

Fig. 1. Regulation of VEGF synthesis by Ras. Activated Ras controls VEGF production at multiple levels of transcription and translation. Rasactivated signal transduction pathways culminate in the phosphorylation of the transcription factors HIF-1, Sp1/3 and AP-2, resulting in enhanced binding to DNA and/or to enhanced transactivation potential. Ras regulation of stress-activated protein kinases may also control mRNA stability by stimulating the binding of proteins to the 3VUTR. In addition, two Ras-regulated pathways stimulate phosphorylation of 4E-BP, leading to release of eIF-4E and to eIF-4E-stimulated initiation of mRNA translation. Finally, wild-type Ras plays a critical role during hypoxia- and acidosisinduced regulation of VEGF synthesis. See text for references and details.

above, making it impossible to present a universal model of Ras-regulated VEGF synthesis and secretion. One or more pathways may predominate under specific conditions in specific cell types and tumors (see for instance Ref. [82]). VEGF synthesis is not only regulated by activated Ras, but also by intratumoral pH and pO2, and by cell density as well as by a number of growth factors. The overall control of VEGF synthesis is therefore determined by the combined signaling pathways activated under these conditions and those that are activated by Ras [82 84]. Is the stimulation of VEGF synthesis essential for Rasstimulated angiogenesis and tumor growth? Grunstein et al. [85] have indeed provided proof of this principle by showing that H-ras-transformed fibroblasts with a targeted deletion of the VEGF gene failed to grow out as fibrosarcomas in mice due to massively reduced vascularity and vascular permeability. In a separate study, Okada et al. [47] showed that down-regulation of VEGF transcription by antisense technology in human colon cancer cells harboring an activated K-ras2 gene greatly reduces their tumorigenic potential in vivo. Conversely, reexpression of VEGF in cells with a deleted K-ras2 oncogene is not sufficient to fully restore tumorigenic potential [47]. Likewise, enforced expression of VEGF in melanomas, initiated by an inducible H-ras oncogene, is not sufficient for maintenance of tumor growth following discontinuation of H-ras expression [27]. In this model system the sustained expression of oncogenic H-ras in the tumor cells is required for maintaining endothelial cell survival and integrity of the tumor vasculature. Surprisingly, endothelial cell apoptosis was observed well before a decline in VEGF synthesis and additional H-ras-

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regulated factors may therefore be critical in maintaining tumor vessel integrity [27]. In conclusion, oncogenic H- and K-Ras stimulate VEGF production and thereby promote tumor vascularisation and tumor growth. Additional Ras-regulated factors are likely to cooperate with VEGF during these processes. Conversely, enhanced VEGF synthesis is not sufficient for either initiation or maintenance of tumor growth in the absence of activated Ras. 2.2. Endogenous Ras controls hypoxia-driven VEGF expression Due to a lack of proper vascularisation and irregular blood flow in the tumor vessels, large areas inside tumors may become hypoxic. Under hypoxic conditions, VEGF production is greatly increased and this is essential for neovascularisation of the hypoxic tissue [40,86]. Interestingly, hypoxia also leads to the activation of Ras [61,87]. Thus, also in tumor types that do not harbor ras oncogenes, wild-type Ras may be activated in hypoxic tumor areas. These findings raise two questions. First, what is the mechanism of hypoxia-induced Ras activation? And second, to what extent does Ras activation contribute to hypoxia-driven VEGF production and angiogenesis? Under hypoxic conditions, reactive oxygen and nitrogen species (ROS and RNS) are formed [88]. Ras can be directly modified and activated by radicals, in particular by SNO [89,90]. Under hypoxic conditions, SNO is synthesized by inducible NO synthase (iNOS), one of the HIFresponsive genes during hypoxia [91]. Through direct Ras activation, SNO may contribute to hypoxia-induced VEGF production. Conversely, SNO has been implicated in the negative regulation of VEGF synthesis through its effects on cGMP signaling [92]. Moreover, whereas Ras activation is robust during hypoxia and modest during reoxygenation [87], radical formation is modest during hypoxia and robust during reoxygenation [88]. These paradoxes need to be resolved in order to assess whether radical modification of Ras can cause or contribute to hypoxia-driven Ras activation. An alternative pathway of Ras activation is that induced by tyrosine kinase signaling, as it occurs after classical stimulation of tyrosine kinase or G protein-coupled receptors. Hypoxia leads to activation of the tyrosine kinase c-Src [93], tyrosine phosphorylation of the adapter protein Shc (a good c-Src substrate), activation of Ras and stimulation of ERK1/2 [61,87]. Interestingly, this signal transduction pathway is similar to the pathway that operates downstream of some (though not all) G-protein-coupled receptors [8]. More work is required to assess whether Src activation is causative in the activation of Ras and ERK1/2 following hypoxia. In addition, it remains unclear how c-Src itself is activated under hypoxic conditions. Is the transient activation of wild-type Ras under hypoxic conditions sufficient to promote VEGF production? Ras

signaling through ERK-mediated activation of the transcription factors Sp1 and Sp3 (presumably through direct phosphorylation [67]) is critical for stimulation of VEGF expression in hypoxic gastric cells [94]. Furthermore, inhibition of Ras signaling (either by using dominant negative Ras or by farnesyltransferase inhibitors (FTIs)) prevents efficient hypoxia-induced VEGF mRNA synthesis and protein secretion in malignant human astrocytoma cells that do not harbor ras mutations [95]. Thus, the induction of VEGF synthesis by hypoxia requires activation of the Ras pathway in addition to stabilization of HIF-1 a (via liberation from VHL). 2.3. Endogenous Ras controls acidosis-driven VEGF expression The intratumoral microenvironment is not only characterized by lower oxygen pressure, but also by a lower pH (acidosis). Recently, it was shown that tumor acidosis induces activation of Ras-dependent signaling to promote VEGF production [96,97]. Thus, two distinct changes in the tumor microenvironment (low pO2 and low pH) independently cause stimulation of Ras signaling and consequent VEGF production.

3. Releasing a break on angiogenesis: Ras represses thrombospondin (TSP) expression Whereas tumor cells expressing activated Ras show elevated levels of VEGF, the levels of thrombospondin-1 (TSP-1) [98 101] and thrombospondin-2 (TSP-2) [100] are dramatically reduced. TSP-1 and TSP-2 are extracellular matrix glycoproteins that are negative regulators of angiogenesis [102 105]. Several mechanism(s) may underlie the anti-angiogenic effects of TSP-1 and TSP-2. Their ability to interact with a variety of ECM components, ECM proteases, cytokines (in particular TGF-h) and growth factors and their direct effects on endothelial cell viability and migration indicate that TSP modulation of angiogenesis is very complex [103,106]. Furthermore, TSP-1 and TSP-2, though sharing their potential to act as anti-angiogenic molecules, do not share complete functional overlap as evidenced by their distinct expression patterns and by the distinct phenotypes of the respective knockout mice [103,107]. Repression of thrombospondin expression was found in cells expressing either one of the three ras isoforms, H-, Kor N-ras [99]. One of the signaling pathways that is switched on by oncogenic Ras (Ras>Rac>MEKK1>JNKK>JNK) leads to phosphorylation of the transcription factor c-Jun, by Jun kinase (JNK). The same pathway causes stimulation of c-jun gene transcription in cells expressing activated Ras [108]. Activated c-Jun, like activated Ras, strongly represses TSP-1 gene expression [98,109]. It seems likely therefore that c-Jun activation by Ras mediates, or at least contributes to, TSP-1 gene repression in Ras-transformed cells.

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In a mouse model for the development of intestinal adenomas, endogenous TSP-1 expression is down-regulated in regions of dysplasia that undergo extensive neovascularisation [110]. Importantly, TSP-1 deficiency promotes the initiation and progression of tumor growth, thus establishing a critical inhibitory role for TSP-1 in the early stages of intestinal tumor development [110]. Although it is clear that TSP-1 and TSP-2 have antiangiogenic and anti-tumorigenic properties, it is less clear to what extent the down-regulation of TSP-1 and/or TSP-2 contributes to the stimulation of angiogenesis in tumor cells expressing activated Ras. The chemical induction of skin carcinomas in mice almost invariably involves acquired mutations in H-ras [17,21]. Hawighorst et al. [111] have used this model to study the functions of TSP-1 and TSP-2 during skin carcinogenesis. They found that the levels of TSP-1 declined to undetectable levels early during tumor development, possibly resulting from activation of H-ras. Conversely, the expression of TSP-2 increased dramatically, but TSP-2 expression was observed in the stromal cells rather than in the tumor cells [111]. Thus, chemically induced skin carcinogenesis is accompanied by decreased TSP-1 production and by enhanced stromal cell production of TSP-2, leading to elevated TSP-2 levels in the tumor matrix. These changes in TSP expression and deposition occur during the very early stages of papilloma development concomitantly with increased expression of VEGF in the tumor cells [111] and, possibly, following ras activation. In the same model, overexpression of TSP-1 in the mouse skin greatly impaired initiation of tumor development and angiogenesis of the dysplasias [112]. Conversely, TSP-2 deficiency greatly facilitated tumor development [111]. Further studies should clarify whether early mutational activation of H-ras is indeed causative in the observed

changes in TSP expression during skin tumor development. If so, the repression of TSP-1 expression is presumably one of the critical early events in Ras-stimulated angiogenesis and tumor development.

4. Ras regulation of the inflammatory modulators cyclooxygenase (COX)-1 and COX-2 4.1. COX-2 controls tumor growth and angiogenesis Chronic inflammation has been associated with the development of various common types of cancer and creates a pro-angiogenic environment [113 115]. In line with this notion, the regular intake of nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin reduces the risk for developing colon cancer by 40-50% [116] and may even cause regression of preexisting adenomas [117]. NSAIDs inhibit cyclooxygenases (COX-1 and COX-2), enzymes that convert arachidonic acid into prostaglandin H2 (PG-H2) which is further converted into inflammatory mediators like PG-F2a, PG-D 2 , PG-E 2 , PG-I 2 and thromboxane A 2 (TX-A 2 ) [118,119]. COX-1 is ubiquitously expressed and controls prostaglandin synthesis under physiological conditions, for instance in the control of renal blood flow and maintenance of the integrity of the gastric mucosa [118]. In contrast, COX-2 expression is inducible by growth factors and oncogenes and is highly expressed in many solid and hematologic tumors, as well as in inflamed tissue. As a result, high PG levels are found under these pathological conditions [118,120]. The importance of elevated COX-2 expression for tumor-associated angiogenesis and tumor progression has been reported in several studies demonstrating inhibitory effects of COX-inhibitors on angiogenesis

Fig. 2. Regulation of COX-2 synthesis by Ras. Activated Ras controls COX-2 production at multiple levels of transcription and translation. Ras-activated signal transduction pathways culminate in the phosphorylation of the transcription factors C/EBP, PEA3 and AP-1, resulting in enhanced transactivation potential. Ras regulation of ERK1/2 and PKB activities may also control mRNA stability by stimulating the binding of proteins to the 3V UTR. Presumably, Ras-regulated pathways also control initiation of COX-2 mRNA translation by stimulating phosphorylation of 4E-BP. Finally, activation of wild-type Ras may contribute to the critical activation of NF-n-B during hypoxia-induced regulation of COX-2 synthesis. See text for references and details.

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and tumor development [115,119,121 123]. In addition, COX-deficient mice are refractory to tumor development in the skin and in the intestines [124 126]. It should be noted, however, that non-COX targets may also be critical for the anti-angiogenic and anti-tumorigenic effects of NSAIDs [127 129]. The following processes that are relevant to tumor outgrowth have been associated with elevated COX-2 levels: (i) increased tumor cell survival; (ii) increased tumor cell invasive capacity; (iii) sustained chronic inflammation; (iv) increased production of toxic and mutagenic molecules through peroxidation of metabolites; and (v) increased tumor-associated angiogenesis [130]. In this section we will only discuss the relevance of elevated COX-2 expression in (Ras-stimulated) angiogenesis. 4.2. Mechanism of COX-2-induced angiogenesis How does increased COX-2 expression, as it is observed in many tumors and pre-malignant lesions, stimulate angiogenesis? At least two distinct COX-2-stimulated processes play an important role. First, it stimulates the production of pro-angiogenic factors like bFGF, PDGF and VEGF [131], and second, it is required for endothelial cell migratory responses through integrin signaling [132]. The product of COX-2 activity, PG-H2, is converted into the biologically active molecules PG-E2, PG-D2, PG-I2, PG-F2a and TX-A2. Although all of these molecules display some form of bioactivity, only TX-A2, PG-I2 and PG-E2 have reported pro-angiogenic effects [133 137]. PGE2 activates a specific class of G protein coupled receptors (EP1-4) and stimulates VEGF and bFGF expression through cAMP signaling [138 141] (Fig. 3). Interestingly, COX-2 synthesis depends on the presence of the PGE2 receptor EP2 [142]. Thus, enhanced PGE2 levels not only cause up-regulation of VEGF and bFGF, but also stimulate COX-2 expression and subsequent PGE2 synthesis in a positive feedback loop. Tumor cell-produced TX-A2, PGI2 and/or PGE2 may stimulate the tumor cells themselves in an autocrine fashion, but they may also activate receptors on nearby endothelial cells, fibroblasts or inflammatory cells in a paracrine fashion. Tumor cells can thus affect the behavior of non-tumor cells in the microenvironment of the tumor. Is COX activity and PGE2 signaling essential for the production of pro-angiogenic factors and the subsequent stimulation of angiogenesis? COX inhibitors indeed prevent the production of VEGF and bFGF by colon cancer cells or fibroblasts [131,143] in in vitro angiogenesis assays, as well as during tumor-associated angiogenesis [131,144]. Furthermore, signaling through EP2 by the COX-generated product PGE2 is essential for VEGF production, angiogenesis and tumor development in the intestine [142,145]. Finally, stromal fibroblasts are a major source of VEGF in the tumor and the expression of both COX-2 and the PGE2 receptor EP3 is essential for VEGF expression by these cells [143,144]. Taken together, COX activity and signaling by its products

are essential for the production of pro-angiogenic growth factors and for the stimulation of angiogenesis in vivo. 4.3. Induction of COX-2 gene expression by Ras COX-2 gene expression is regulated by a variety of signals that are involved in either initiation or promotion of tumor growth: (i) deregulation of the Wnt pathway [146,147]; (ii) loss of p53 expression [148]; (iii) HER-2/neu overexpression [149]; (iv) hypoxia [150]; and (v) signaling by oncogenic Ras and Src [151 154]. Distinct Ras-activated signaling pathways have been implicated in the control of COX-2 expression (Fig. 2). First, the Ras>Rac>MEKK1>JNKK>JNK pathway promotes COX-2 transcription through the cyclic-AMP response element (CRE) via phosphorylation of c-Jun, a component of the transcription factor activator protein 1 (AP1) [155]. In addition, the classical Ras>Raf >MEK>ERK1/2 pathway is required for COX-2 up-regulation by activated H-ras [152] presumably through phosphorylation of the CCAAT/enhancer binding protein h (C/EBP h) [156] and/or the Ets transcription factor PEA3 [149]. H-Rasstimulated phosphorylation of PEA3 is mediated by both MAPK and JNK [157]. PEA-3 activation is also essential for the stimulation of uPA gene expression by oncogenic Ras (see below) [158,159]. The stability of the 3VUTR of COX-2 mRNA is greatly increased by expression of activated K-ras2 in intestinal epithelial cells, a phenomenon that is caused by activation of both the Ras>Raf >MEK>ERK1/2 pathway as well as the Ras >PI(3)K>PDK>PKB pathway [160]. The latter pathway also controls the efficiency of cap-dependent mRNA translation [73]. However, it is not known whether activated Ras stimulates the initiation of COX-2 mRNA translation, as it does with the VEGF mRNA (see above). Deregulated Wnt-signaling occurs early in colon cancer development following inactivation of APC. As a result, the transcription factor TCF is de-repressed and TCF-mediated signaling ensues. A functional TCF-binding promoter element (TBE) in the COX-2 promoter may contribute to enhanced COX-2 expression following APC gene inactivation [147]. In addition, activation of the Wnt pathway leads to up-regulation of PEA3 and to PEA3-mediated COX-2 transcription [161]. Taken together, both Wnt- and K-Rasstimulated pathways cooperate in the stimulation of COX-2 expression and this may be especially relevant during the development of early neoplasias in the colon (Fig. 2). 4.4. The contribution of COX-2 to Ras-stimulated angiogenesis Mutations in Ras are initiating events in tumor development and can be detected in pre-malignant tissues. Similarly, COX-2 overexpression is detected at the very early stages of tumor development in pre-malignant hyperplasias in the pancreas, the colon and the lung (associated with K-ras2

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Fig. 3. Up-regulation of COX-2 and COX-initiated signaling in response to Ras. The up-regulation of COX-2 in tumors harboring activated Ras leads to the enhanced production of prostaglandins. PGE2, PGI2 and thromboxane A2 have reported pro-angiogenic effects. Stimulation of PGE2 receptors EP2 or EP3 either on the tumor cells themselves or on stromal cells leads to increased synthesis of cAMP and to cAMP-stimulated VEGF (and possibly bFGF) gene transcription in both tumor and stromal cells. EP receptor activation also leads to further stimulation of COX-2 synthesis, thereby creating a positive feedback loop. See text for references and details.

mutations) and in the bone marrow (associated with N-ras mutations) [119,162]. Although activated Ras certainly contributes to COX-2 expression (see above), other inputs can also regulate COX-2 expression, such as hypoxia [163], cytokines and growth factors [164]. Interestingly, COX-1 and COX-2 are dispensable for Hras-induced transformation of fibroblasts in vitro [128]. Unfortunately, the ability of COX-1- or COX-2-deficient cells to grow as fibrosarcomas in vivo was not further investigated in this study. During initiation of tumor growth, the up-regulation of these enzymes by activated Ras presumably affects processes other than initial cellular transformation. Obviously, the stimulation of angiogenesis is one such process. The initiation and progression of chemically induced skin carcinomas in the mouse is invariably accompanied by mutations in H-ras [21,165] and is greatly reduced in COX-1- and COX-2-deficient mice [125], as it is in Hras-deficient mice [165]. This suggests that COX-1 and COX-2 play an essential role in H-Ras-initiated skin tumorigenesis. However, the reduced tumorigenesis in both COX1- and COX-2-deficient animals is presumably due to the premature terminal differentiation of keratinocytes [125]. A wealth of data highlight the essential role for COX-2, PG-E2 and the PG-E2 receptors EP2 and EP3 in the stimulation of angiogenesis and tumor development in mice [124,142 145]. However, these studies do not provide proof for the involvement of COX-2 and its products in Ras-induced angiogenesis. With the development of mouse models in which tumorigenesis can be induced by conditionally switching on activated ras alleles [166,167], it should be feasible to study the contribution of COX-2 and PG-E2-EP2/ 3 signaling to Ras-initiated tumor development and angiogenesis. Given the central role for COX-2 in mediating

inflammatory responses and the fact that chronic inflammation is a pro-angiogenic condition [113 115], the stimulation of COX-2 expression by Ras will undoubtedly contribute to Ras-stimulated angiogenesis.

5. Changes in the proteolytic environment induced by Ras Activated Ras stimulates the expression of several proteases that break down the extracellular matrix. These include urokinase plasminogen activator (uPA) [168,169] and matrix metalloproteases (MMP-2 and MMP-9) [170, 171], proteases that play a critical role in the control of tumor angiogenesis [172]. Overexpression of uPA is associated with poor prognosis and enhanced tumor dissemination in several types of cancer [173]. uPA is one of the two proteases (the other being tissue-type plasminogen activator, tPA) that controls conversion of the inactive zymogen plasminogen into the active protease plasmin. Plasmin is a broad specificity serine protease. Its classical function is to degrade fibrin in blood clots (fibrinolysis), but other substrates include components of the extracellular matrix and of basement membranes. Plasmin also activates matrix metalloproteases, resulting in further breakdown of the ECM [174]. The uPA receptor (uPAR) serves to localize plasmin formation to the cell surface [175], thereby facilitating tumor cell migration through extracellular matrices and basement membranes [176]. In addition to directing cellassociated plasmin formation, uPA activates intracellular signaling pathways through the uPAR, thereby stimulating cell proliferation and migration in a manner that is independent of uPA proteolytic activity [177].

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Activated Ras stimulates uPA expression through distinct elements in the uPA gene promoter. First, a combined PEA3/AP1 binding motif at 1967 binds Ets transcription factors (targets for the Ras>Raf >MEK>ERK1/2 pathway) and c-Jun (a target for the Ras>Rac>MEKK1>JNKK>JNK pathway) [158,159]. An additional AP1 motif at 1880 has also been implicated in the Ras-dependent stimulation of uPA expression [178]. In addition to stimulating the classical effector pathways that lead to MAPK and JNK activation, oncogenic Ras also directly activates guanine nucleotide exchange factors for the small GTPase Ral [179]. Ral-dependent signaling is critical for stimulation of uPA expression (but not MMP-2 or MMP-9 expression) by Ras [180], as evidenced by expression of a dominant negative Ral mutant. It is presently unknown how Ral signaling affects uPA promoter activity. Activated Ras also stimulates expression of the matrix metalloproteases MMP-2 and MMP-9. Multiple elements in the MMP-9 promoter, including PEA3- and AP1-binding sites, are essential for promoter stimulation by H-Ras [170]. Thus, optimal stimulation of both uPA and MMP-9 gene expression by Ras requires signaling through MAPK- and JNK-responsive promoter elements. Conversely, stimulation of the MMP-9 promoter by activated H-Ras in kidney epithelial cells depends on the intracellular generation of radical oxygen species and subsequent activation of NF-n-B [181]. In addition to activating transcription factors that act on the MMP-9 promoter to stimulate gene transcription, Ras and/or Myc also stimulates translation of the MMP-9 mRNA in prostate carcinoma cells [182]. As discussed above in Section 2.1, Ras may enhance cap-dependent mRNA translation by disrupting the complex between 4E-BP and eIF-4E through stimulation of 4E-BP phosphorylation. The uPA uPAR complex on endothelial cells may promote the migration of developing sprouts through the provisional matrix that is primarily composed of fibrin [183]. This process is essential during the early phases of angiogenesis. Likewise, enhanced production of uPA (and other proteases) by tumor cells enhances tumor cell migration and invasion. Obviously, enhanced uPA expression in endothelial cells cannot be the direct consequence of Ras activation in tumor cells. This raises the question whether the elevated levels of uPA that are produced by tumor cells contribute to the stimulation of angiogenesis. Two distinct mechanisms should be considered. First, plasmin and MMPs can liberate pro-angiogenic growth factors like VEGF and bFGF from the ECM, thereby making them available for endothelial cell activation [184 187]. In fact, liberation of VEGF by MMP-9 (produced by infiltrating inflammatory cells) controls the onset of angiogenesis in oncogene (SV40-largeT)-induced pancreatic tumors [188]. In addition, the latent form of the pro-angiogenic cytokine TGF-h can be converted into active TGF-h by plasmin [186]. Signaling by activated TGF-h may subsequently synergize with Ras signaling in the induction of VEGF synthesis [84].

Highly vascularised melanomas can be induced in mice by application of specific carcinogens [189]. The majority of chemically induced skin tumors harbor an activated Hras oncogene [21] and activation of Ras (or Ras effector systems) is critical for melanoma development in the mouse [190]. Interestingly, uPA-deficient mice are refractory to melanoma induction by carcinogens [191]. Reduced (plasmin-mediated) liberation of pro-angiogenic growth factors may underlie this phenomenon [191]. Nevertheless, formal proof for the involvement of MMP-9, MMP-2, and/or uPA in the stimulation of angiogenesis by tumor cells expressing activated Ras is lacking. Second, protease-generated degradation products of ECM components themselves may have positive or negative effects on endothelial cell migration, proliferation and angiogenesis [172]. In fact, protease-generated fragments from plasminogen, collagens IV, XV and XVIII possess strong anti-angiogenic and anti-tumor activity in mouse models through mechanisms that are still largely unclear [172]. Many of these biological compounds are now being further tested in clinical trials. It remains to be established to what extent protease-generated ECM fragments positively or negatively control angiogenesis during Ras-induced tumorigenesis. Taken together, oncogenic Ras promotes tumor angiogenesis by at least three independent mechanisms: (i) by stimulating the production of pro-angiogenic growth factors; (ii) by reducing the production of anti-angiogenic factors; and (iii) by ensuring the availability of pro-angiogenic growth factors through local degradation of the ECM (Fig. 4).

Fig. 4. Ras-stimulated pathways that promote angiogenesis. Oncogenic Ras up-regulates VEGF expression in tumor cells, but can also do so in nearby stromal cells through stimulation of COX-2 expression, subsequent prostaglandin synthesis and activation of stromal cell PG receptors (EP). In a parallel pathway, oncogenic Ras stimulates the production of uPA and MMPs that break down the extracellular matrix surrounding the tumor cells. As a result, growth factors are liberated and become available for endothelial cell activation. Moreover, ECM breakdown products themselves directly affect endothelial cell behavior and angiogenesis (positively or negatively). Activated Ras also represses expression of the antiangiogenic matrix glycoprotein TSP-1. See text for references and details.

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6. Isoprenylation inhibitors as anti-angiogenic compounds Posttranslational prenylation is required for the association of all Ras proteins (H-Ras, N-Ras, K-Ras2-4A, K-Ras24B) with the plasma membrane. The cellular precursor for prenylation is 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), which is converted into mevalonate by HMG-CoA reductase. Mevalonate is further metabolized into farnesylpyrophosphate and, subsequently, into geranylgeranylpyrophosphate. The latter two molecules can be covalently attached to a large variety of proteins, including Ras, by protein:farnesyl transferase (PFT) and protein:geranylgeranyl transferase (PGGT). The C-terminal CAAX motif is a target for either farnesylation (H-, K-, and NRas) or, when PFT is inhibited, geranylgeranylation (K- and N-Ras). In addition, K-Ras2-4B has a C-terminal lysine-rich region (positively charged) that allows electrostatic interaction with the (negatively charged) plasma membrane. Since farnesylation is critical for proper Ras localization and, hence, for Ras function, FTIs as well as HMG-CoA inhibitors (statins) have been extensively studied for their potential anti-tumor effects. Some of these compounds have shown promising results in in vitro experiments, in tumor models in mice, as well as in clinical trials. Interestingly, growing evidence suggests that both classes of compounds have profound anti-angiogenic effects in vitro as well as in vivo [192 197]. Treatment of tumor cells with these compounds is accompanied by decreased VEGF secretion [195 200], possibly due to reduced Ras signaling. However, the results from phase I clinical trials show that also tumors that do not harbor activated Ras oncogenes can respond to FTI treatment [201,202]. In the absence of activated mutant Ras, tumor growth and VEGF secretion may still require signaling through wild-type Ras. Such tumors may therefore still be FTI-sensitive [95,203]. Nevertheless, it is becoming increasingly clear that even though they were developed as specific Ras inhibitors, inhibition of tumor growth by FTIs and statins may rely on the inhibition of targets other than Ras. Many proteins aside from Ras are isoprenylated, and some of these are involved in cellular signal transduction and cell cycle control themselves [204,205]. In addition to affecting tumor cell behavior, FTIs and statins also inhibit endothelial cell migration and tube formation in vitro [192 194,197]. Again, inhibition of Ras signaling may not always underlie the observed effects [192]. Thus, FTIs and/or statins may interfere with tumor angiogenesis directly through inhibitory effects on endothelial cell activation, and indirectly by lowering VEGF secretion by tumor cells. Taken together, the antitumor activity of prenylationinhibiting compounds is presumably due to inhibition of more than one target and to modulation of multiple biological processes within the tumor. Nevertheless, Ras activity plays a crucial role in the control of tumor angiogenesis and

it seems likely therefore that the inhibition of angiogenesis by these compounds is due, at least in part, to inhibition of Ras function.

Acknowledgements The authors thank Drs. Hans Bos and Pat Crowley for critically reading the manuscript and for their helpful comments.

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