Angiogenesis as a strategic target for prostate cancer therapy

Angiogenesis as a StrategicTarget forProstate CancerTherapy

Yong Li1,2 and Paul J. Cozzi2,3

1Cancer Care Centre, St George Hospital, Sydney, NSW 2217, Australia2Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia

3Department of Surgery, St George Hospital, Sydney, NSW 2217, Australia

Published online 18 June 2009 in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/med.20161

.

Abstract: It is becoming increasingly clear that angiogenesis plays a crucial role in prostate cancer

(CaP) survival, progression, and metastasis. Tumor angiogenesis is a hallmark of advanced cancers and

an attractive treatment target in multiple solid tumors. By understanding the molecular basis of

resistance to androgen withdrawal and chemotherapy in CaP, the rational design of targeted therapeutics

is possible. This review summarizes the recent advancements that have improved our understanding of

the role of angiogenesis in CaP metastasis and the potential therapeutic efficacy of inhibiting angio-

genesis in this disease. Current therapeutic options for patients with metastatic hormone-refractory CaP

are very limited. Targeting vasculature is a developing area, which shows promise for the control of late

stage and recurrent CaP disease and for overcoming drug resistance. We discuss angiogenesis and its

postulated mechanisms and focus on the regulation of angiogenesis in CaP progression and the ther-

apeutic beneficial effects associated with targeting of the CaP vasculature to overcome the resistance to

current treatments and CaP recurrence. & 2009 Wiley Periodicals, Inc. Med Res Rev, 30, No. 1, 23–66, 2010

Key words: angiogenesis; metastasis; antivascular agents; prostate cancer

1. INTRODUCTION

Prostate cancer (CaP) is the most common malignancy and is the second leading cause ofcancer death in males.1 In 2008 alone, it was estimated that CaP would be diagnosed in1,863,202 men, and 28,660 would die of the disease.2 Most patients will present with localizeddisease. For this group of men, therapeutic options include radical prostatectomy (RP),radiotherapy, and active surveillance.3 RP is one of the most common treatments for CaPand generally provides excellent cancer control. However, approximately 35% of patientswill develop a prostate-specific antigen (PSA) recurrence (metastatic disease) within 10 yearsafter surgery.4,5 Studies indicate that several clinical parameters (PSA doubling time,

Contract grant sponsors: Australian Research Council (ARC); Cancer Institute NSW.

Correspondence to: Yong Li, Cancer Care Centre, St George Hospital, Gray St Kogarah, Sydney, NSW 2217, Australia, E-mail:

[email protected]

Medicinal Research Reviews,Vol. 30,No.1, 23--66, 2010

& 2009 Wiley Periodicals, Inc.

pathological Gleason score, and time from surgery to biochemical recurrence) help predictthe outcomes of men with PSA elevation and risk stratify patients for CaP-specific mortalityfollowing biochemical recurrence after RP.6–9 The standard therapy for locally advanced ormetastatic disease is androgen deprivation therapy via surgical or medical castration. Theinitial therapeutic response to the anti-androgen-therapy is only brief (8 month–3 years), andCaP patients then become refractory to additional treatment, as tumors eventually relapse toan androgen-independent (AI) state10 and the development of castrate-resistant prostatecancer (CRPC), which no longer responds to androgen ablation. Docetaxel was shown toprolong survival in patients with CRPC,11,12 but chemotherapeutic strategies for effectivedisease control are still required. Despite demonstrating an improvement in overall survival,responses are not durable and eventual progression of disease is inevitable. Alternativetreatment strategies and novel approaches are needed. One such strategy is angiogenesisinhibition.

Angiogenesis plays a critical role in CaP progression, and its significance in human CaPhas been firmly established.13,14 Several independent studies have documented a significantcorrelation between microvessel density (MVD) with Gleason score, pathological stage, andpatient survival.13,14 In contrast, another study indicated that CaP angiogenesis correlateswith progression after RP but not with pathologic stage in Gleason sum 5–7 adenocarcinomaof the prostate.15 These data indicate the patients from different subpopulations may result indifferent results. Furthermore vascular endothelial growth factor (VEGF) levels are sig-nificantly increased in CaPs (relative to normal tissue), an upregulation that directly corre-lates with tumor stage, differentiation, and disease-specific survival.16 Serum VEGF levels aresignificantly higher in metastatic CaP patients compared with localized disease.17 Hypoxic-inducible factor (HIF), a key mediator of VEGF expression, is highly expressed in CaP,compared with the normal prostate and benign prostate tissue.18 Veltri et al. have recentlydemonstrated Her-2/neu oncogene and nuclear roundness variance are significant in theprediction of progression-free survival and suggested integration of image analysis-basednuclear roundness variance and molecular biomarkers with pathologic parameters should beconsidered for validation in the prediction of progression-free survival.19

The growth of tumor metastases, like that of the primary tumor, requires angiogenesis.Tumors shed millions of cells into blood and lymphatic circulation in a process that requirespenetration through a multilayer barrier composed of pericytes, base membrane, and en-dothelial cells. The ‘‘seed and soil’’ hypothesis posits that tumor cells that have entered thecirculatory system need to extravasate and colonize at predetermined locations.20 Bonemarrow-derived, VEGF receptor (VEGFR)-1-positive progenitor cells are a critical factor inthe assembly of the premetastatic niche, where VEGFR-2-positive endothelial progenitorcells may also be involved.21 The clinical fate of a CaP patient is ultimately determined by theprimary tumor’s capacity to grow, invade locally, evoke angiogenesis, and, eventually, me-tastasize. Bubendorf et al. reported metastatic patterns of CaP from autopsy study of 1,589patients and found hematogeneous metastases were present in 35% of 1,589 patients withCaP, with most frequent involvement being bone (90%), lung (46%), liver (25%), pleura(21%), and adrenals (13%).22 Unfortunately, the mechanisms responsible for this phenom-enon remain largely undefined. Recent evidence, however, suggests a significant role of thetumor cell’s microenvironment, and that bone cells and bone-specific matrix provides apermissive, dynamic context for the formation of metastases.23 Most commonly, humanCaPs metastasize to bone.24,25 Basic and clinical studies indicate that suppression of angio-genesis can inhibit tumor progression and metastasis.26 In this review, we summarize the roleand regulation of angiogenic factors in CaP progression and the potential therapeutic sig-nificance in control of late stage, CRPC disease to overcome the resistance to currenttreatments and CaP recurrence.

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Medicinal Research Reviews DOI 10.1002/med

2. ANGIOGENESIS IN CANCER PROGRESSION

Angiogenesis, the development of new blood vessels, is a fundamental physiological processthat promotes embryonic development, tissue repair, and fertility, yet that also promoteschronic inflammation, tumor growth, and tumor metastasis.27 It involves a complex series ofevents including classic sprouting angiogenesis, the loss of pericyte–endothelial cell adhesion,increased permeability, vasodilation, and the incorporation of bone marrow-derivedendothelial progenitor cells.28–32 Upon binding to membrane receptors in vascularendothelial cells, a five-step process is triggered (Fig. 1): initially the vascular endothelialbasement membrane of the parent vessel breaks down, allowing a route for the developmentof a new capillary sprout, this is followed by migration of endothelial cells through thebasement membrane toward the angiogenic stimulus; this leading front of migrating cells isdriven by enhanced proliferation of endothelial cells, followed by the formation of capillarytubes via organization of the endothelial cells, and a recruitment of periendothelial cells(pericytes) and vascular smooth muscle cells for new capillary stabilization.33

Angiogenesis is defined as the process of forming new blood vessels to support tissuegrowth. Tumors in particular rely on angiogenesis for their continued growth. In fact, it hasbeen demonstrated that solid tumors will not grow larger than 2–3mm in diameter in theabsence of new blood vessels, and require angiogenesis to form metastases.34–36 This step hasbeen termed the ‘‘angiogenic switch’’ (Fig. 1) when tumors acquire the ability to recruit theirown blood supply.37 The process of angiogenesis is mediated by a multitude of pro- and

Angiogenic factors

TumorA

Angiogenesis

B

C

D

Basement membrane break down Endothelial cell

proliferation and migration

Structural recognition and cell-cell adhesion

Figure 1. Angiogenesis and the progression of tumor growth. An angiogenic switch results in a tumor with an angiogenic

factor-secretingphenotype (A). Angiogenic factors cause thepermeabilizationandbreak-downof thebasementmembrane in the

surrounding vasculature (B). Endothelial cells are stimulated to proliferate and migrate toward the angiogenic factor-secreting tu-

mor (C). Microvascular endothelial networks composed of loosely connected cellular cords differentiate into capillary tubules.

Structural reorganization and penetration of the capillary sprouts into the tumor provides blood flow to the growing tumor (D).[Color figure canbe viewed in the online issue, which is available at www.interscience.wiley.com.]

ANGIOGENESIS AS A STRATEGICTARGET K 25


anti-angiogenic factors, which will be discussed in the following section. In tumors, activationof the angiogenic switch signifies a shift in the balance toward pro-angiogenic factors.38

Animal studies suggest that this occurs early in cancer development and is the rate-limitingstep for progression.39 Furthermore, the tumor’s ability to activate angiogenesis is a pre-requisite for metastatic spread. An angiogenic phenotype has been associated frequently witha poorer prognosis in many cancers.37

New blood vessels in tumors can grow by sprouting from preexisting vessels or by re-cruitment of rare, circulating bone marrow-derived endothelial progenitor cells and mono-cytes.27,40,41 Circulating bone marrow-derived cells (BMDCs, monocytes) migrate into tumorsin response to tumor-secreted chemokines and differentiate into macrophages.Tumor cells, macrophages, and fibroblasts within tumors can secrete a number of potentpro-angiogenic cytokines, such as VEGF, tumor necrosis factor a (TNFa), interleukin 8 (IL8),and basic fibroblast growth factor (bFGF, also known as FGF2)41–43 and express a broadarray of extracellular matrix (ECM)-degrading proteases, including urokinase-type plasmi-nogen activator (uPA), the matrix metalloproteinases (MMPs) including MMP2, MMP7,MMP9, and MMP12, and elastase.42 IL8 expression correlates with the angiogenesis, tu-morigenicity, and metastasis of tumors in numerous xenograft and orthotopic in vivo models.44

Recently, IL8 signaling has been implicated in regulating the transcriptionalactivity of the androgen receptor (AR), underpinning the transition to an AI proliferation ofCaP cells.45 Importantly, new evidence suggests that macrophage infiltration can activate theangiogenic switch in spontaneous tumors.43 Selected integrins have key roles in regulating thetrafficking of circulating monocytes and progenitor cells to tumors.46 The role of BMDCs intumor neovascularization is currently the subject of intense research and debate. Evidence forthe existence of endothelial progenitors has come from studies demonstrating the ability ofBMDCs to incorporate into adult vasculature. However, the exact nature of endothelialprogenitor cells remains controversial.47 Because of the lack of definitive markers ofendothelial progenitors, the in vivo contribution of progenitor cells to physiological and pa-thological neovascularization remains unclear. Numerous studies support the view that cells ofthe hematopoietic system contribute to tumor angiogenesis. However, there is considerablecontroversy regarding the identity and function of such cells. Using C57BL/6 mouse model,Ziegelhoeffer et al. reported in the adult organism, BMDCs do not promote vascular growthby incorporating into vessel walls but may function as supporting cells.48

In another study, Shinde Patil et al. further demonstrated bone marrow-derived lin�c-kit1Sca-11 stem cells do not contribute to vasculogenesis in lewis lung carcinoma.49 Someinvestigators hold the view that the major contribution of the bone marrow to angiogenesis isthrough stem cell-like endothelial progenitor cells, which are able to differentiate in tumorendothelium. Other lines of investigation instead point to the conclusion that the differentia-tion of bone marrow cells into endothelium is a very rare event if it occurs at all, and that theproduction of angiogenic factors by tumor-infiltrating inflammatory cells is the major me-chanism.50 The details of angiogenic regulation will be discussed in the following section.

Tumor heterogeneity leads to heterogeneity in the tumor vasculature. Just as there aremultiple phenotypes for any given tumor type, so can there be multiple phenotypes of thetumor angiogenic process. The onset of angiogenesis, or the ‘‘angiogenic switch,’’ is a discretestep that can occur at any stage of tumor progression. It depends on the type of tumor and itsmicroenvironment.

Conditions in the tumor cell microenvironment, hypoxia for example, trigger theangiogensis cascade. Increased levels of hypoxia, as measured in CaP patients, are directlycorrelated with raised levels of expression of VEGF.51 Huss et al.52 performedimmunohistochemistry and in situ analyses of tissue specimens using a transgenic mousemodel to investigate the relationship between HIF-1, VEGF, and angiogenesis. They

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suggested that there were two distinct angiogenic events involved in the development andprogression of CaP. The first angiogenic ‘‘initiation switch’’ is associated with increasedexpression of HIF-1 and VEGFR plus recruitment and amplification of intraductal vascu-lature.52 The second angiogenic ‘‘progression switch’’ is associated with an increased level ofexpression of VEGF protein in prostatic tissues as well as in the sera of mice harboringadvanced, poorly differentiated, AI tumors.52 This implies that the abnormally raisedexpression of HIF-1 protein in CaP cells may have the ability to make these cells moreaggressive via mechanisms of both androgen resistance and angiogenesis promotion.Secretion of proteolytic enzymes, including MMPs and uPA, lead to degradation ofbasement membranes and allow endothelial cells to migrate and organize themselves intopericyte-supported tubules.53 These so-called ‘‘capillary sprouts’’ subsequently form luminaand connect via anastomoses, followed by the re-synthesis of basement membrane.37,54 A keyfeature of these new tumor vessels is that they are structurally abnormal and differ in theirbehavior from normal blood vessels, which include aberrant vascular structure, alteredendothelial cell–pericyte interactions, abnormal blood flow, increased permeability, anddelayed maturation.38 Such ‘‘leaky’’ and inefficient tumor vessels deliver less blood, oxygen,nutrients, and ultimately anticancer drugs to the tumor, increasing hypoxic conditions andthereby keeping the angiogenesis cascade perpetually active.38

3. POSTULATED MECHANISMS OF ANGIOGENESIS IN CaP METASTASIS

In an attempt to support their own metabolic needs, malignant tumors typically induceneovascularization of themselves by evoking the influx of endothelial cells. These cells, inturn, proliferate and differentiate into new blood vessels. The mechanisms that control theseevents in prostate carcinogenesis have been under close scrutiny, as knowledge of the specificevents hold potential for the design of clinical treatments for both early- and later-stageCaPs.

It is clear that the microenvironment of a CaP includes ‘‘reactive stroma,’’ comprisingmultiple cell types, which have been altered from their original (normal) state to becomepermissive of CaP cell progression. In human CaPs, reactive stroma is characterized by anincrease in myofibroblasts, a corresponding amplification in ECM protein production, andan increase in local vascular density.55

Tumor blood vessels exhibit characteristic markers that are not present in normalangiogenic tissues.56 After enduring the circulation ‘‘journey,’’ metastatic cancer cells canescape out of the endothelial vasculature and into the target tissue via extravasation. How themetastastic cells signal activating changes in the vascular permeability of blood vessels intarget organs is a complex process involving several factors. VEGF, initially identified aspotent vascular permeability factor, is the lead candidate. Activation of Src family kinases inendothelial cells exposed to VEGF induces disruptions in endothelial cell junctions,facilitating metastatic extravasation. Hypoxia within the tumor mass applies selective pres-sure promoting the outgrowth of malignant cells, with diminished apoptotic ability. Thecellular response to low oxygen tension involves stabilization of a HIF-1 transcriptionalcomplex gene involved in cell survival and invasion. In the prostate, VEGF is secreted byboth epithelial cells and smooth muscle cells.57 West et al. reported that VEGF expression inCaPs correlated to PSA levels and Gleason score.58 CaPs remain dormant and clinicallyundetectable until they begin to secrete angiogenic factors and downregulate the expressionof angiogenic inhibitors,59 a defined early event in tumor development known as theangiogenic switch.60 The result is an imbalance of angiogenic factors leading to growth ofnew vessels and the tumor.



4. LYMPHOGENESIS IN CaP METASTASIS

The lymphatic vasculature is an important route for the metastatic spread of human cancer.It was previously thought that lymphatic metastasis involved passage of malignant cells alongpreexisting lymphatic vessels near a tumor; however, recent studies in animal models suggestthat lymphangiogenesis can be induced by solid tumors and may promote tumor spread.61

The major lymphangiogenic cytokines are VEGF-C and VEGF-D. VEGF-C and -D, thebona fide ligands for VEGFR-3, belong to the VEGF family of angiogenic factors.62

By binding to VEGFR-3, which is predominantly expressed on lymphatic endothelial cells,they induce the formation of new lymphatic vessels (lymphangiogenesis). Levels of VEGF-C/D have generally correlated with lymph node metastasis in human patients.63 Platelet-derivedgrowth factors (PDGFs)64 and VEGF-A65 are also implicated in tumor lymphangiogenesis.However, the evidence for involvement of these molecules in tumor lymphangiogenesis iscurrently restricted to relatively few animal models and therefore requires analysis in abroader range of experimental models as well as extensive clinicopathological studies tocorrelate the expression of these molecules with metastasis in human cancer.

Expression of VEGF-C or -D by tumor cells induces lymphatic vessel formation aroundthe tumors and promotes metastatic tumor spreading at draining lymph nodes and distantorgans. While it is clear that lymphatic vessels facilitate metastatic tumor spreading, the exactmechanism by which this occurs has not been fully elucidated.66 Until recently, analysis ofthe role of lymphatics in tumor growth and metastasis had been hindered by the absenceof lymphatic markers. Recent identification of specific lymphatic markers, such as thetranscription factor PROX1 and the CD44 homolog lymphatic vessel hyaluronan receptor 1(LYVE1)67,68 has made it possible to study mechanisms regulating lymphangiogenesis.

In CaP, Tsurusaki et al.69 found that VEGF-C mRNA levels were significantly higher inlymph node-positive tumors and that VEGFR-3-positive vessels were increased in the stromaof VEGF-C-positive tumors. Moreover, VEGFR-3 expression was correlated with Gleasonscore, preoperative PSA levels, and lymph node metastasis in CaP.70 Zeng et al. demon-strated both VEGF-C and VEGF-D are widely expressed in human CaP, and VEGF-D, butnot VEGF-C, is also abundantly expressed in adjacent benign prostate epithelia, andVEGFR-3 is up-regulated in vessels in a subset of CaPs, which is involved in lymphaticmetastasis.71 The study from the same group further provided strong evidence thattumor-induced activation of host lymphatic endothelial cells via VEGFR-2 signalingunderlies CaP lymphatic metastasis.72 Wong recently confirmed that tumor-secreted VEGF-C and, to a lesser extent, VEGF-A, are important for inducing CaP intratumoral lym-phangiogenesis but are unnecessary for lymph node metastasis.73 These data support theVEGF-C/VEGF-D/VEGF-3 (VEGFR-2) signaling pathways may provide targets forantilymphangiogenic therapy in CaP.

5. MVD IN CaP

It has long been observed that CaPs possess a significantly higher number of microvesselsthan the corresponding benign tissue of RPs from patients.74,75 While a positive correlationwas seen between MVD and Gleason score, this was primarily seen in the more poorlydifferentiated tissue typical of a higher Gleason score.75 Several retrospective studies haveshown that mean MVD correlates with increasing Gleason score and disease progression(from extraprostatic extension to metastasis) in CaP.76,77 More recent analysis, however, hasshown a potential for using MVD counts to predict tumor progression from high-gradeintraepithelial neoplasia (HGPIN) to CaP,78 as well as outcome in patients undergoing RP.14

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Concato et al. demonstrated Bcl-2 and MVD are independent predictors of subsequent deathamong men with CaP and might have a clinical role in assisting in deciding on treatment.79

Using computerized whole slide quantification, van Niekerk et al. showed significantdifferences between the vascular bed in CaP tumor tissue as compared with normal prostatetissue and mean and 75th percentile of MVD, vascular area, and vascular perimeter wereincreased in tumor, showing enhanced vascularity.80

The monoclonal antibody (MAb) CD34 has been used to visualize microvessels, and ap-pears to be a reliable method for obtaining an MVD count.14,81,82 Assessing the amount ofvasculature present in both precancerous and cancerous lesions is a strategy that may help todetermine both the type and aggressiveness of treatments to employ in curing patients of CaP.Future assessment of MVD could be useful in the clinical trials using anti-angiogenic therapies.

6. ANGIOGENIC FACTORS IN CaP METASTASIS

Angiogenic stimuli produced due to metabolic demands of host tissues initiate the angiogenicresponse.83 In normal conditions angiogenesis is maintained by an intricate balance betweenendogenous stimulators of angiogenesis and endogenous inhibitors of angiogenesis. Duringtumorigenesis, the angiogenic switch is activated directly via induction of angiogenic growthfactors, or indirectly, by recruiting host immune cells that release mediators of angiogen-esis.59 Circulating endothelial precursor cells from the bone marrow (BMDCs) may alsocontribute to tumor neovascalurization,40 while tumor cells can recruit new blood vessels dueto a network from adjacent endothelial cells,84 although there may have different opinionsabout the development of angiogenesis as discussed above. The stimulators of angiogenesis inCaP are summarized in Table 1. The regulation of angiogenesis by different stimulators inCaP progression is shown in Figure 2.

Table 1. Stimulators of Angiogenesis in Prostate Cancer Metastasis

Stimulators References

Hypoxic-inducible factor-a (HIF-a) Elucidated in the review

Vascular endothelial growth factor (VEGF) Elucidated in the review

Prostate specific membrane antigen (PSMA) Elucidated in the review

Insulin-like growth factor-1 (IGF-1) Elucidated in the review

Extracellular matrix metalloproteinase inducer protein Elucidated in the review

(EMMPRIN/CD147)

Matrix metalloproteinases (MMPs) Elucidated in the review

Androgen Elucidated in the review

Integrins Elucidated in the review

Endothelins Elucidated in the review

Angiopoietins Elucidated in the review

Urokinase plasminogen activator (uPA) 85

Basic fibroblast growth factor (FGF2) 86

Transforming growth factor-beta (TGF-b) 33,87

Platelet-derived endothelial cell growth factor (PD-ECGF) 88,89

Cyclooxygenase-2 (COX-2) 90–92

Hepatocyte growth factor (HGF) 93,94

Interleukin-8 (IL-8) 95–97

MUC1 98



A. HIF-a

Tumors require a blood supply to grow more than 2–3mm in diameter.34 However, thevessels generated have an abnormal structure, are leaky and demonstrate blood flowcharacteristics such that tumors are frequently stressed by hypoxia.99 This microenviron-mental pressure results in upregulation of HIF, one of the basic helix loop helix-PAS familyof transcription factors.100 Three a subunits, such as HIF-1a, HIF-2a, and HIF-3a, havebeen identified. Under normoxia, prolyl-hydroxylation of the HIF-a subunit is enhanced,and mediates interaction with von Hippel–Lindau protein (pVHL), E3 ubiquitin ligase, thatthen undergoes proteosomal degradation. At low oxygen levels, prolyl-hydroxylation of HIFis inhibited, allowing it to escape degradation through pVHL. HIF-a translocates to thenucleus to form a heterodimeric complex with HIF-b and subsequently stimulates genetranscription.101 HIF induces transcriptional activation of genes regulating key processes intumor progression, such as angiogenesis (Ang-2), glucose metabolism, (glucose transporter 1),adhesion (E-cadherin, Vimentin), migration (TGF-a, c-Met), proteolysis (Cathepsin D, uPAreceptor, MMP2), and pH (CA IX; carbon anhydorase 9).102 Tumors that exhibit abundantHIF-1 stabilization have a greater likelihood of developing metastatic relapse and result in

Figure 2. Mechanisms regulating angiogenesis in CaP metastasis. (A) Tumor cells near preexisting blood vessels secrete

growth factors and chemokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor

necrosis factor a (TNFa), hepatocyte growth factor (HGF), and urokinase plasminogen activator (uPA), that stimulate quiescent

vascular endothelium to enter the cell cycle. (B) These growth factors activate or upregulate the expression of prostate specific

membrane antigen (PSMA), VEGF, integrins, endothelin, Angiopoietin, and platelet-derived endothelial cell growth factor

(PDECGF). (C) These stimulators then promote endothelial cell migration and survival during invasion of tumor tissue, resulting inthe creation of new vessel sprouts.The new blood vessels promote tumorgrowth by removing waste products and providing nu-

trients.These new blood vessels also provide anavenue for tumor metastasis. (D) Angiogenesis promotesmetastasis to local anddistant sites, such as bone. [Color figure canbe viewed in the online issue, which is available at www.interscience.wiley.com.]

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shorter survival with a subset of HIF target genes acting as mediators of metastastic pro-gression.103

Hypoxia in human tumors has long been linked to radioresistance.104 Hypoxia has alsobeen shown to be a potent stimulus for tumor progression, mediated via effects onangiogenesis, genetic instability, glycolysis, inhibition of apoptosis, and upregulation ofgrowth factors.105,106 Transcription of the angiogenic factor VEGF is directly controlled byHIF-1a. Huss et al.107 demonstrated that the early angiogenic ‘‘initiation switch’’ correlatesexpression of HIF1-a and VEGFR-1 in addition to the recruitment and elaboration ofintraductal vasculature in prostatic intraepithelial neoplasia (PIN) lesions in transgenicadenocarcinoma of the mouse prostate (TRAMP) model. Increased expression of HIF-1amRNA in rat CaP cell lines and hypoxia-induced expression of HIF-la protein in human CaPcell lines have been shown to be associated with increased cell growth rates and metastaticpotential.108 Acute hypoxia has also been shown to result in increased aggressiveness andsurvival of LNCaP cells, a localized CaP line known to be noninvasive in vivo.109

The accumulation of HIF-1a protein has been previously reported to be an early event inCaP and in HGPIN.110,111 Although the number of prostate specimens examined waslimited, HIFs appear to be upregulated through transition from normal, hyperplasia,prostatic PIN to invasive carcinoma.101,112 Monsef et al.113 have recently further confirmedthe expression of HIF-1a and HIF-2a in CaP and also identified immunoreactive HIF-1aand downstream gene products in benign and malignant prostate neuroendocrine cells.

Overexpression of HIF-1a has been shown to stimulate the invasive potency of humanCaP cells through the epithelial–mesenchymal transition (EMT) pathway.114 The expressionof VEGF in CaP is associated with the extent of tumor hypoxia,115 with both HIF-1a116 andVEGF117 being overexpressed in CaP tissues in comparison with benign prostatic tissues. InCaPs HIF expression has also been correlated with expression of AR levels118,119 leading tothe suggestion that both HIF-1a and HIF-2a have a role in this disease process. Boddyet al.119 hypothesized that androgens may regulate VEGF levels through the activation ofHIF in androgen-sensitive tumors, and that inhibition of both HIF pathways may providenew therapeutic options in the management of this disease. Muir et al.120 have suggested thathypoxia triggers a physiologically relevant increase in VEGF-A in CaP cells and bonemarrow stromal cells, which involves a paracrine loop that recruits and activates bonemarrow endothelium to support tumor neovascularization-related processes. Vergis et al.121

have recently demonstrated that increased expression of HIF-1a and VEGF were each sig-nificant predictors of worse freedom from biochemical failure, independent of T stage,Gleason score, initial PSA concentration, and each other. These findings are consistent withthe data available on the role of tumor hypoxia and angiogenesis in progression of CaP.

B. VEGF

The VEGF family of proteins consists so far of seven known ligands called VEGF A–E andprostaglandins F 1/2.122–124 VEGF-A is commonly known as VEGF and in humans occurs inat least five splice variants.37–125 VEGF signals primarily via a transmembrane tyrosinekinase receptor called VEGFR-2 (synonymous with KDR or Flk-1) and induces most of itseffects via this route, including endothelial cell proliferation and survival.35,36,124,126 VEGF-Aalso binds to VEGFR-1 (Flt-1) and neuropilin, a coreceptor present on endothelial, stromal,and tumor cells; VEGF-A both enhances and regulates VEGFR-2 signaling. VEGF-B onlybinds to VEGFR-1, while VEGF-C and VEGF-D bind to both VEGFR-2 and VEGFR-3(Flt-4) and are involved in lymphangiogenesis.124,127

VEGF stimulates vascular endothelial cell proliferation and endothelial cell survival128

as well as the secretion of proteolytic enzymes (MMPs, uPA), which break down ECM.129,130



Its paracrine and autocrine signaling loops promote tumor cell growth and survival.131,132

Tumor cells and stromal cells, like fibroblasts, produce VEGF.133 Inhibition of the VEGFpathway markedly decreases angiogenesis and inhibits the growth of tumors in nude mouseexperiments.134,135

VEGF is the most prominent regulator of physiological angiogenesis.136 It initially in-teracts with VEGFR-2 to promote endothelial cell proliferation, migration, and vascularpermeability, and subsequently with VEGF-R1 to assist the organization of new capillarytubes. VEGFR-1 is expressed mostly in vascular endothelial cells and is important in thetrafficking of inflammatory cells,137 whereas VEGFR-2 is expressed mainly in endothelialcells and is responsible for recruiting cells in the developing vasculature,138 induction ofMMPs139 and secretion of additional growth factors from the developing endothelium.140

Loss of VEGFR-1 impairs the ability of angioblasts to be organized into mature capillariesin vivo.141 VEGFR-3 preferentially binds VEGF-C and VEGF-D and its expression in theadult is primarily on lymphatic endothelial cells. More recent data has demonstrated theexpression and function of VEGFR-3 on vascular endothelial cells.142 The details ofVEGFR-3, VEGF-C/D in regulation of lymphangiogenesis have been discussed in Secton 4.VEGFR3 has a crucial role in postnatal lymphangiogenesis.143,144 These VEGFRs are tyr-osine kinases that function in cell survival, motility, and proliferation via different down-stream pathways involving phospholipase C, calcium, and PI3K/Akt, and the RAS/RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)pathways.145 The diverse VEGF isoforms and their ability to heterodimerize together effectthe complicated tissue-specific regulation of proliferation and survival.

VEGF promotes tumor angiogenesis, and hence blood flow through several mechanisms,including enhanced endothelial cell proliferation and survival, increased migration andinvasion of endothelial cells, increased permeability of existing vessels, forming a latticenetwork for endothelial cell migration, and enhanced chemotaxis and homing of bonemarrow-derived vascular precursor cells (both endothelial cells and pericytes).146,147

CaP cells express VEGF in vitro and in vivo.148 VEGF expression by CaP specimens149

and CaP cell lines (LNCaP, PC-3, and DU 145) is far greater than that by stromal cells of thenormal prostate.150,151 VEGF expression may also contribute to CaP-induced osteoblasticactivity in vivo.152 Using a TRAMP model, Isayeva et al. demonstrated that inhibitors of theVEGFR-2 delayed tumor progression only when administered in the early stages of CaP,before a significant rise in VEGF levels was observed. This same inhibitor was ineffective ifadministered during the later stages of CaP, when VEGF levels were high.153 Thus, theminimal effects on tumor progression in clinical trials of angiogenesis inhibitors may be dueto the advanced stage of CaP being targeted.

Clinical studies comparing CaP with benign prostate hyperplasis (BPH) revealed thatVEGF expression was correlated with increased levels of angiogenesis, and that this was atleast partly mediated by VEGF.154 The levels of VEGF in serum, plasma, or urine arecorrelated with patient outcome in both localized as well as disseminated CaP.17,155,156

In addition, the levels of the VEGFR were correlated with a poorer grade of tumordifferentiation and prognosis in CaP.107

Normal prostate tissue expresses minimal to no VEGF,157 unlike CaP tissue that stainspositively for VEGF in areas of increased MVD.151 A correlation between elevated serumVEGF levels and CRPC was discovered, which suggests the potential clinical usefulness forVEGF as a prognostic factor in CaP.156 Of interest, recent studies have shown that VEGFexpression is stimulated in the presence of androgens. In this regard, androgen ablationtherapy is a clinical means of suppressing tumor angiogenesis in vivo.158 In a study of 50patients with locally advanced disease treated with radical radiotherapy, Green and co-workers reported a correlation between higher VEGF expression and worse disease-specific

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survival (p5 0.035).159 Furthermore, Peyromaure et al. compared 17 patients who developedbone metastases after RP with 23 patients who remained disease free and found the expressionof VEGF-A was significantly higher in those who developed bone metastases after RP.160

Higher plasma concentrations of VEGF have also been associated with biochemicalprogression after RP.161 These results suggest VEGR plays an important role in CaP me-tastasis and could be useful as a target for novel therapy.

C. PSMA

PSMA is a type two transmembrane protein with a short intracellular amino terminal of 18amino acids, a transmembrane region of 24 amino acids, and a glycosylated extracellulardomain of 706 amino acids,162 which has folate hydrolase or carboxy peptidase II activity.When the cDNA and gene encoding PSMA were cloned, and the exon/intron structure of thegene determined, it was renamed as folate hydrolase 1 (FOLH1).162,163 It is also called asglutamate carboxypeptidase II (GCPII) and is a target enzyme for diagnosis and treatment ofCaP. PSMA shares homology with the transferrin receptor164 and is internalized with similarkinetics as the transferrin receptor.165 Several features of this molecule suggest that PSMA isa multifunctional protein; however, a defined role of PSMA in cancer is still not known.166

In the prostate, several variants of this protein were found, among them so-called PSM0,which lacks the first 59 N-terminal amino acids and is dominant (on the mRNA level) innormal human prostate, while full-length PSMA is the prevalent form in primary prostatetumors.167–169 PSMA/GCPII expression is also up- or downregulated in some nonprostatictumors.170

PSMA was originally identified in epithelial cells of prostatic origin.171 Its expression isincreased several fold in malignant disease, with the greatest levels observed in advanced CaPand AI disease.172,173 The full-length, transmembrane form of PSMA is dramatically upre-gulated in the majority of advanced CaPs.167 In these tumors, there is a strong correlationbetween a negative prognosis and cell surface expression of PSMA.174,175 PSMA is absent inmost other tissues and cell types, including normal endothelial cells and preformed bloodvessels; however, expression has been detected in the angiogenic sprouts of tumor-associatedneovasculature for most solid cancers examined.172,176,177 PSMA is associated with tumorvasculature, including nonprostate tumors, but not normal vasculature.177 Anilkumar et al.demonstrated that PSMA binds to caveolin-1 and undergoes internalization via a caveolae-dependent mechanism, and they hypothesize that it is possible that PSMA, filamin A, andcaveolin-1 are in a complex that might be involved in the regulation of signaling eventsassociated with the formation of neovasculature in tumors.178 Conway et al. showed thatangiogenesis is severely impaired in PSMA-null animals and that this angiogenic defectoccurs at the level of endothelial cell invasion through the ECM barrier.179

The endothelial cells of tumor-associated neovasculature, not the tumor cells, containedPSMA mRNA transcripts.180,181 These results strongly support the hypothesis that theendothelial cells of tumor-associated neovasculature synthesize the PSMA protein and do notsequester the protein from the serum or the surrounding stromal cells. These findings implythat the PSMA promoter and PSMA gene or the surrounding sequence contains tran-scriptional enhancer regions that selectively activate PSMA transcription in tumorneovasculature but not in normal vessels. The selective expression of PSMA intumor-associated angiogenic endothelial cells raises the intriguing possibility that PSMAmight have a functional role in expansion, remodeling, or maintenance of the neovascularnetwork. Its function in the prostate remains unclear, its specific expression on angiogenicvasculature suggests that it participates in neovessel growth in developing tumors, a rolesimilar to those of other cell surface peptidases with analogous vascular expression



patterns.181 Moreover, it is upregulated in the vasculature of most solid tumors and istherefore a potential target for the generation of novel antineoplastics. Its precise con-tribution to prostate tumorigenesis is currently under investigation. Recently, Pomper’sgroup have demonstrated selective imaging of xenografts that express PSMA using smallanimal positron emission tomography and single photon emission computed tomographyand the urea-based PSMA inhibitors for CaP diagnosis.182–184 These approaches are muchbetter than J591 MAb imaging. They further developed the low-molecular weight,urea-based inhibitors incorporating tridentate chelators for binding of the fMðCOÞ3g 1core,(M) 99mTc, 186,188Re), while retaining high affinity to PSMA as a imaging probe.185 Be-cause of high stability and favorable labeling characteristics, the organometallicRe(I)(CO)3/99mTc(I)(CO)3 approach represents an attractive radiolabeling strategy forfuture CaP imaging.

D. IGF-1

The IGF family involves the combination of two ligands (IGF-I and IGF-II), two receptors(IGF-IR and IGF-IIR), six high-affinity binding proteins (IGFBPs 1–6), a large group ofIGFBP proteases, and a new group of proteins, low-affinity IGFBP-related proteins.Members of this family form a network of interactions both among themselves and withother growth factor families and their signaling pathways.186 IGF-I is a 70 amino acidpeptide and its gene is located on chromosome 12. IGF-II is a 67 amino acid peptide and itsgene is located on chromosome 11 close to H19, a paternally imprinted gene. Loss of IGF-IIimprinting has been described in several tumors. This loss of imprinting leads tooverexpression of this growth factor. Its precise role in tumorigenesis and tumor growth isnot clear.187 IGF-I is mainly produced by the liver under hypothalamic growth hormone-releasing hormone and pituitary growth hormone control. Like IGF-II, but in contrast toinsulin, IGF-I can be synthesized by almost every tissue in the body. Thus, IGFs areproduced in most organs and exert biological effects on most cell types. Despite its presenceat higher concentration in the circulation, IGF-II is believed to play a less important rolethan IGF-I in postnatal growth.

IGF-I has been implicated in tumor angiogenesis through stimulation of VEGF.188,189

It can regulate the growth and function of normal and malignant prostate cells.190 IGF-Ibelongs to a family of related proteins whose bioactivity, both in the circulation and intissues, is modulated by a number of binding proteins, of which IGFBP-3 has been mostclosely associated with an inhibition of prostate carcinogenesis.191,192 Nearly 90% of IGF-Icirculating in serum is bound to IGFBP-3, which is also primarily produced in the liver.192

Although primarily synthesized in the liver, IGFs are produced locally by most tissues inwhich they act in an autocrine or paracrine manner.191,193,194 IGF-I binds to the IGF-Ireceptor, a tyrosine kinase receptor that transduces signals to the nucleus and mitochondrionprimarily via the MAPK and PI3k/Akt pathways.195,196 In addition to direct contributions toeach of these stages, IGF-I may promote cancer indirectly, through interactions withoncogenes and tumor suppressors, interactions with other hormones, and interactions withthe IGF binding proteins.193,195,197

IGF-1 is a potent stimulator of cellular proliferation and survival as well as tumorgrowth. While serum levels of IGF decrease with age, within the aged population thoseindividuals with the highest levels of serum IGF-1 have the greatest risk of developingepithelial cancers such as CaP.198 During progression of CaP, local levels of both IGF-1 andits receptor (IGF-1R) increase.198 Like many growth factors, IGF-1 has the potentialto reverse the age-associated decline in endothelial cell function.199 Moreover, IGF-1upregulates the expression of modulators of endothelial cell function such as VEGF,

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membrane type1 (MT1)-MMP, and MMP2; this regulation requires signaling through theIGF-1R via both the PI3K and MAPK pathways, which modulate endothelial cell functionand subsequent angiogenesis.197,200,201 Goel et al. reported that interactions between theIGF-1R and b1 integrins also activated signaling through both the PI3K and MAPKpathways, which resulted in enhanced CaP cell migration on and invasion through theECM.202

Recent in vitro studies suggest that IGF-I may act in a paracrine and autocrine fashionto promote CaP cell proliferation and survival.191,192,203,204 In TRAMP mice, IGF-I has beenimplicated as an important factor in the development and progression of CaP.198,205,206

Elevated IGF-I levels in the TRAMP model have also been proposed to induce theexpression of VEGF, thereby facilitating angiogenesis and leading to metastatic spread of thedisease.198 In this TRAMP model, serum IGF-I levels correlate with the increase in meanvessel density associated with the development of HGPIN lesions, suggesting a relationshipbetween IGF-I and the induction of prostatic neovascularization.198

Several large epidemiological studies showed that higher concentrations of IGF-I andlower concentrations of IGFBP-3 are associated with a higher risk of CaP.207,208 Increasedlevels of IGF-I was found in men with CaP compared with men with BPH orcontrols.198,203,209,210 This high level of IGF could contribute to the initiation and progres-sion of CaP because it is known to induce VEGF.198 It has been proposed that IGF isinvolved in the angiogenic switch leading to prostatic neovascularization.198 The IGF axis isan important regulator of growth and development and changes in IGF signaling haveimportant implications in malignant growth of CaP.191,193,194,198,209,211 Deregulation of theIGF axis and elevated serum levels of IGF-I are associated with CaP.194,197,198,212 Theseresults indicate that IGF-I is an important stimulator in CaP angiogenesis.

E. EMMPRIN

EMMPRIN is a variably glycosylated multifunctional transmembrane glycoproteinexpressed widely, especially on tumor cells, with potential for homophilic binding andinteractions with integrins, cyclophilins, annexin II, and caveolin-1.213 EMMPRIN canstimulate MMP expression by tumor cells, stromal fibroblasts, and endothelial cells214

through direct cell-to-cell contact, or indirectly by shedding of whole protein inmicrovesicles,213 or by proteolytic shedding.215 Highly glycosylated EMMPRIN inducesMMP activation, although the signaling pathways are not well defined.213 High levels ofEMMPRIN are reported in numerous malignancies, including CaP,216 and are associatedwith cancer progression.217 Transcriptome analysis and comparative genomic hybridizationof individual tumor cells isolated from bone marrow of CaP patients showed thatEMMPRIN is the most frequently expressed protein in primary tumors and inmicrometastases.218 Furthermore, knockdown of EMMPRIN expression with smallinterfering RNA in PC-3 CaP cells decreased invasiveness and reduced MMP-2 and MMP-9secretion in vitro.219 EMMPRIN and MMPs are considered significant prognostic factors inhuman CaP.220 These results suggest that EMMPRIN plays an important role in CaPmetastasis.

EMMPRIN can stimulate expression of MMP-1, MMP-2, and MMP-3 in endothelialcells,221 and induces their expression, together with MT1-MMP and MT2-MMP, in fibro-blasts.222 Secreted and transmembrane MMPs are important in ECM remodeling and play amajor role in tumor invasion and metastasis, including roles in angiogenesis, inflammation,apoptosis, cell surface protein-shedding, release and activation of ECM-sequestered growthfactors such as TGF-b and FGF2, and signal transduction.223 Using MDA-MB-231 humanbreast cancer cells engineered to express different levels of EMMPRIN, Tang et al. have



shown that EMMPRIN can mediate angiogenesis via stimulation of vascular VEGFsynthesis.224 Millimaggi et al.225 demonstrated that transfection of EMMPRIN/CD147cDNA into the CABA I ovarian cancer cell line enabled CABA I-derived vesicles to induceangiogenesis and to promote MMP gene expression in human umbilical vein endothelialcells, suggesting that vesicles shed by ovarian cancer cells may induce pro-angiogenicactivities of human umbilical vein endothelial cells via a CD147-mediated mechanism.Tumor cell anchorage-independent growth, a characteristic of malignant cancer cells that iscritical for metastatic spread, can also be potentiated by EMMPRIN.226,227 In mammarycarcinoma cells, EMMPRIN-stimulated production of hyaluronan is associated with tumormalignancy and multidrug resistance.228 Interaction of hyaluronan with cell surfacereceptors can induce anchorage-independent growth, involving suppression of anoikis(loss-of-anchorage cell death), through inhibition of the pro-apoptotic protein Bim.226 Therole(s) of EMMPRIN in CaP survival, anchorage-independent growth and angiogenesisremain to be explored.

F. MMPs

MMPs are a family consisting of 16 members of zinc-dependent proteases229 that mediateECM degradation. The four major subgroups of this protease family are gelatinases,collagenases, stromelysins, and membrane-associated proteases.230 Most MMPs released intheir inactive state are cleaved to activation by other MMPs or other serine proteases such asplasmin and uPA.231,232 Tissue inhibitors of matrix metalloproteinases (TI-MMPs) providean additional level of regulation for MMP activation.231 ECM undergoes constantremodeling in normal homeostasis and MMPs function to remove proteins from thebasement membrane during this remodeling.229 Their cooperation targets degradation ofbasement membrane of the vascular endothelium and ECM, thus creating a passageway inthese physical barriers toward new capillary formation.233

The influence of MMPs on tumor propagation results from both direct and indirectmechanisms.234 Direct effects, via degradation of the matrix, result in a more permissiveenvironment for cell migration and invasion. The subsequent facilitation of the angiogenicresponse results in a greater blood supply to the tumor. Indirect effects of MMP activityinclude activation of other pro-MMPs, cleavage of regulatory precursor molecules at the cellsurface, and induction of nascent chemokines and growth factors that require enzymaticactivation. MMP activity also has been shown to be a key component of VEGF-inducedangiogenesis in tumors,235 reflecting another pathway by which MMPs interact withcomponents of the ECM to facilitate vessel formation and tumor growth.

CaPs show high levels of gelatinase (MMP2 and MMP9) expression and activity236 thatis related with higher-grade and increased metastatic potential.237 Primary and metastaticCaP display heterogeneous MT1-MMP expression, reported to be involved in CaP migrationvia cleavage of laminins and collagen I.238,239 High levels of plasma and urine MMP-2 andMMP-9 in CaP patients have been found to correlate with CaP metastasis.240,241 Inhibitionof MMP-9 by ribozyme technology or synthetic MMP inhibitors reduces the metastaticpotential and invasiveness of CaP cells in mice.242 Antisense inhibition of MMP-9 can alsoattenuate angiogenesis, human CaP cell invasion, and tumorigenicity in a mouse xenograftmodel.243 These data support the role of MMPs as stimulators of CaP angiogenesis.

G. Androgen

Androgens may regulate the vasculature both in experimental and human CaPs. Afterandrogen ablation of nude mice implanted with the androgen-sensitive human CaP LNCaP,endothelial cells begin to undergo apoptosis before neoplastic cells, and involution of the

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tumor vessels precedes the decrease in tumor size.244 When blood flow was measured in theprostate of androgen ablated CaP patients, the first response was a reduction in blood flowpreceding a reduction in tumor volume.245 Moreover, a six-fold increase in prostateendothelial cell apoptosis246 and a decreased prostate vascular permeability247 was reportedin patients with CaPs treated with androgen ablation. These results suggest that androgensare indeed involved in the regulation of the vasculature and CaP growth is angiogenesisdependent.

Although serum androgen levels decrease with age, the levels of active androgen in theprostate, dihydrotestosterone (DHT), do not decrease due to increased activity of the5a-reductase enzyme that converts testosterone to DHT.248 In CaP epithelium, androgenscan stimulate angiogenesis.249 Following androgen withdrawal in AI tumors, there is adecrease in angiogenesis associated with tumor regression. However, there is invariably areturn of tumor that is castration-resistant.250 These tumors are commonly referred to as AI,although castration-resistant may be a more appropriate term as they still contain significantlevels of testosterone and DHT.250 In castration-resistant tumors, there is an increase inangiogenesis that is associated with an increase in MMP-9.243 These studies indicate thattumor angiogenesis in CaP is associated first with androgens, then with an increase in matrixremodeling proteases. Further, these data imply that anti-angiogenic drugs should be ofpotential therapeutic benefit. However, no definitive clinical trials have been published,which may reflect a unique ability of CaP to bypass the usual age-associated inhibition ofangiogenesis.

Androgens have also been implicated in the upregulation of HIF-1a expression inCaP.251 Previous in vitro studies of CaP cells have demonstrated that angiogenesis isinfluenced by androgens, and that this effect is partly mediated by HIF.158 Androgendeprivation has been shown to decrease angiogenesis in CaP in animals158 and improvetumor oxygenation in patients with CaP.252 Richard et al. deciphered an androgen-mediatedmechanism of VEGF modulation in a mouse prostate model.57 Their findings show that, inluminal epithelial cells and in smooth muscle cells of the prostate, the secretion of VEGF iscontrolled by androgens. As the luminal cells secrete VEGF apically, it is probable that theandrogen/VEGF-mediated angiogenesis observed in the prostate (and in CaPs) is primarilydue to the stromal smooth muscle.57 The androgen/VEGF axis described by these workersmay lend further credence to the notion that the stromal microenvironment criticallyinfluences the growth and progression of CaPs.

H. Integrins

Integrins are transmembrane proteins that serve a role as primary mediators of cell–ECMinteractions that are functionally involved in determining tumor angiogenic responses duringcancer progression to metastatic disease. Integrins (heterodimers containing two distinctchains, a and b subunits), recognize the major adhesive ECM components, fibronectin andlaminin, leading to regulation of cell proliferation, cell survival, anoikis, and migration.253,254

Angiogenic growth factors such as VEGF and pFGF can exert a profound positive effect onthe activity and expression of several integrins, such as avb3, avb5, avb1, a3b1, a3b1, a6b1,a6b4.255 Additional angigogenic factors, such as class 3 semaphorins (SEMA3 proteins),control vascular remodeling via targeting integrin.256 Increased expression of integrin avb3 isdetected in growth factor-activated endothelial cells in tumor blood vessels and granulationtissue,257,258 while very low expression has been detected in resting blood vessels.259

Expression of the avb3 in tumor endothelium correlated with the aggressive phenotype inneuroblastoma.260 Moreover, blocking integrin avb3 with a MAb-mediated endothelial cellapoptosis and inhibited blood vessel formation,257 implicating its functional significance in



angiogenesis. Integrins also contribute to signal transduction from the extracelluarenvironment to the intracellular network mediated by integrin-activated signaling molecules,such as focal adhesion kinase, PI3K, and members of the ERK1 and 2/MAPK ( family toregulate cell proliferation, migration, and apoptosis of tumor and endothelial cells.261,262

Angiogenesis, a process critical for tumor formation and growth is regulated by integrinfunctions.263 Both avb3 and avb5 regulate angiogenesis by cross-talking with growth factorsignaling pathways.264 Several studies report deregulation of integrin expression as CaPprogresses to an advanced stage.24,261,265 Most a and b subunits have been shown to bedownregulated in CaP.266 Using severe combined immunodeficiency-human-bone model,Nemeth et al. demonstrated that inhibition of avb3 integrin reduces angiogenesis, boneturnover, and tumor cell proliferation in experimental CaP bone metastases.267

Sun et al. have evaluated the efficacy of a new camptothecin conjugate, JF-10-81, ananti-angiogenic agent, in a CaP mouse model.268 JF-10-81 blocks cancer cell adhesion in vivoand angiogenesis in C57BL/N mice and reduces the expression of avb3 and avb5 on PC-3cells, which implies an inhibitory effect on angiogenesis through an avb3- and avb5-dependent mechanism. Mahabeleshwar et al. found that a knock-in mouse expressing amutant b3 that cannot undergo tyrosine phosphorylation shows that b3-deficient mice haveimpaired capillary formation in response to VEGF stimulation, and thus form smallerprostate tumors than their wild-type counterparts.269 These observations further confirm therole of vascular avb3 in CaP through modulation of angiogenesis.269

I. Endothelins (ETs)

ETs, a class of proteins involved in vasoconstriction, have also been linked to angiogenesis.Epithelial cells produce three types of endothelins, ET-1, 2, and 3270 that interact with tworeceptors, endothelin A receptor (ETAR) and endothelin B receptor (ETBR). ET-3 stimu-lates ETBR to induce endothelial cell growth and blood vessel formation.271 A dynamiccross-talk between ET-1 and ETAR promotes VEGF release leading to a strong angiogenicresponse.272,273 ET-1 produced by cancer cells acting via ETAR and ETBR has been linkedto neovascularization, both in the tumor as well as in the surrounding vasculature.270 The ETaxis has a relevant role in various cancer cells and stromal cells leading to autocrine/paracrineloops that activate aberrant proliferation, escape from apoptosis, new vessel formation,immune modulation, abnormal osteogenesis, alteration of nociceptive stimuli, invasion, andmetastatic dissemination.274,275

Elevated expression of ET-1 and its cognate receptor is significantly associated withMVD and VEGF expression in tumor cells. Thus, ET-1 increases VEGF mRNA expressionand induces VEGF levels in a time- and dose-dependent fashion, and does so to a greaterextent under hypoxia.275 Under hypoxic conditions, ET-1 potentiates hypoxia stimulus byamplifying HIF-1a stability and VEGF production.276

In the normal prostate gland, ET-1 is produced by epithelial cells; the highestconcentrations of ET-1 are found in seminal fluid. In CaP, key components of the ET-1clearance pathway, ETBR and neutral endopeptidase, are diminished, resulting in an increasein local ET-1 concentrations.275 Increased ETAR expression is also seen with advancingtumor stage and grade in both primary and metastatic CaP.277 By activating ETAR, ET-1 ispathogenically involved in facilitating several aspects of CaP progression, includingproliferation, escape from apoptosis, new bone formation, or altering the equilibrium in painmodulation.275

The various mechanisms by which ET-1 modulates angiogenesis include endothelial cellproliferation, migration, invasion, protease production, tube formation, and the productionof VEGF. VEGF is a potent stimulant of angiogenesis and works at least in part by inducing

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endothelial cell proliferation and vascular permeability by increasing the levels ofHIF-1a.272,274,278,279 VEGF is overexpressed in many tumors, including CaP, and levels havebeen correlated with CaP stage, grade, and clinical outcome.280 In vivo, the combination ofET-1 and VEGF produced significantly more angiogenesis than either alone.270,272 Thus, dueto the increased levels and interplay between ET-1 and VEGF, targeting the ET axis appearsto be a rational approach to preventing or decreasing neovascularization and thus CaPprogression.

J. Angiopoetins (Ang)

Ang-1 and Ang-2 have both been identified as ligands for tie-2 receptor (Tie-2), a receptorexpressed on endothelial cells.281,282 It has been previously shown that Ang-1 and Ang-2 playcritical roles in angiogenesis, in concert with VEGF.283 Ang-1 binding to Tie-2 maintains andstabilizes mature vessels by promoting interaction(s) between endothelial cells andsurrounding ECM. Ang-2, however, competitively binds to Tie-2, and antagonizes thestabilizing ability of Ang-1, resulting in an overall destabilization of vessels.

Ang-1 and its antagonist Ang-2 act via the Tie-2 and are important regulators of the laterphases of angiogenesis. In most tissues they act together with VEGF to form a fullyfunctional vasculature.284,285 Several studies have implicated Ang in tumor growth andprognosis, often in concert with VEGF.286,287 The role of the angiopoietins in the prostateand in CaPs, is however largely unknown. Studies on human and mouse tissues have shownthat Ang-1, Ang-2, and Tie-2 are expressed in the prostate on both RNA and proteinlevels.288,289 Ang-2 expression was significantly related to histological grade, vascular density,metastases, and to cancer-specific survival.290 Using cDNA microarrays to compare geneexpression in CaP bone vs. liver and lymph node metastases, Morrissey et al. reportedAng-2 is central in regulating the Tie-2 and vessel remodeling in CaP metastases. However,Satoh et al. demonstrated Ang-1 could also induce angiogenesis as well as the maturation ofblood vessels in PC-3 tumors, resulting in the enhancement of tumor growth.291 Caine et al.found a significant decrease in Ang-1 and Ang-2, VEGF after RP in CaP patients.292

Differential expression of Ang-2 and VEGF in AI CaP models, which indicates differentroles of these factors in the regulation of angiogenesis in different stages of CaP.293 Thesedata support both Ang-1 and Ang-2 that have close relationship with VEGF and make thempossible targets for future therapies.

6. INHIBITING ANGIOGENESIS IN CaP THERAPY

Anti-angiogenesis is a promising therapy for CaP, and several anti-angiogenic agents arecurrently under investigation in clinical trials.294–297 In contrast to other conventionaltherapies that destroy tumor cells directly, anti-angiogenic therapy is directed specificallyagainst microvascular endothelial cells that have been recruited into the tumor bed.298 Thus,anti-angiogenic therapy does not induce acquired drug resistance.299 As tumor growth islargely limited by the degree of neovascularization, many clinical strategies have evolved toantagonize the process of angiogenesis and specifically target nascent microvascularendothelial cells.300 The endothelial cells of tumor-associated neovasculature differ in manysubtle ways from their normal vascular counterparts.301

Therapeutic strategies include the delivery of endogenous inhibitors of angiogenesis,agents that prevent the degradation of the basement membrane or ECM agents that interferewith or block the action of pro-angiogenic factors, and small molecule inhibitors of angio-genic factor receptors found in CaP. The aim of anti-angiogenic therapy is to transform CaP



to a chronic disease state. Several mechanisms for inhibiting angiogenesis have been used,including blocking of endothelial cell proliferation, blocking angiogenic factors that areinvolved in cell proliferation such as VEGF and FGF by neutralizing antibodies, or affectingdownstream pathways by receptor kinase inhibition.302

Anti-angiogenic therapy is a promising adjuvant to conventional therapies that shouldhelp to overcome their current limitations and enhance their antitumor effects.303 The precisemechanism by which angiogenesis inhibition improves clinical outcome is not yetunderstood. On the one hand, anti-angiogenic therapy targets endothelial cells rather thancancer cells and the resulting loss of tumor vasculature limits the nutrient supply to the tumorand inhibits growth or induces apoptosis in surrounding cancer cells.304 On the other hand,evidence suggests that excess VEGF by itself causes poor blood flow in disorganized andleaky tumor vessels, resulting in increased interstitial fluid pressure, and accordingly poordrug delivery and hypoxia. Jain et al. suggested that anti-VEFG therapy ‘‘normalizes’’ bloodflow thereby increasing the delivery of oxygen as well as chemotherapy.31 Preclinical andclinical evidence shows that anti-angiogenic agents enhance chemotherapeutic efficacy305 andother data suggest that this also may be the case for synergism with radiotherapy.306 Thedetails concerning inhibition of angiogenesis in CaP will be discussed in the following section.

A. Inhibiting Angiogenesis By MAb

1. MAb J591PSMA is qualitatively distinct from other neovascular targets such as VEGF, endoglin, orintegrins that are involved in the general process of angiogenesis and are not specific to tumorvasculature. PSMA expression has not been reported in normal vasculature and PSMArepresents the only qualitatively specific neovascular target currently known. This specificitymakes PSMA an ideal target for the delivery of a cytotoxic agent designed to destroy tumorvasculature. The development of antibodies to the extracellular domain of PSMA(PSMAext) led to the clinical study of one anti-PSMAext MAb J591.176 The human re-combinant MAb J591 (MLN591) was generated by Dr. Neal Bander and is being developedby the BZL Company. This antibody has been genetically modified to replace certain regionsof the mouse protein with human sequences that are more likely to reduce the possibility of ahuman antimouse antibody response, or human antimouse antibody (HAMA) reaction.

MLN591 has been employed as a naked antibody, radioconjugate, or chemoconjugate.Two phase I trials using radiolabeled MAb J591 in patients with advanced CaP demon-strated acceptable toxicity, excellent targeting of known sites of CaP metastases, and biologicactivity.307,308 Using 111Indium (111In) labeled MAb J591 as a vascular-targeting agent,Milowsky et al.309 demonstrated that MAb J591 specifically targets PSMA on the vascularendothelium of metastases in patients with multiple types of solid tumors and with minimaltoxicity, and confirmed that PSMA is a valid target for vascular-targeting agent therapy.Vascular-targeting agents represent a promising therapeutic approach to the management ofsolid tumor malignancies, with the anti-PSMA MAb J591 demonstrating excellent targetingof metastatic sites in multiple advanced solid tumors without significant toxicity. Futuretrials should further evaluate the dose and schedule using MAb J591 as a naked antibodyalone or in combination with chemotherapy and as a targeted delivery system for a cytotoxicagents or radioisotopes. In addition, these trials should explore the use of PSMAimmunohistochemistry and/or imaging as a basis for patient selection for therapy and the useof appropriate end points for vascular targeted therapies.

2. BevasizumabBevacizumab (Avastin; Genentech, South San Francisco, CA, USA, and Roche, Basel,Switzerland) is a humanized MAb that binds the VEGF ligand and prevents receptor binding

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and signal transduction.310 Since March 2008, bevacizumab has been approved for treatingpatients with late-stage colon cancer, nonsmall-cell lung cancer and breast cancer, all incombination with chemotherapy.

This agent has shown promising activity both in preclinical and in phase I and II studiesand is well tolerated compared with conventional cytotoxic chemotherapy.311,312 However,Bevacizumab as a single agent has been studied in CaP with no significant objective re-sponses. In a phase II study using bevacizumab 10mg/kg every 14 days for 6 infusions, nopatients had a PSA decline 450% and only 4 of 15 patients achieved a PSA decline ofo50%, with no definite objective or partial responses noted after one cycle.313 Anotherphase-II Cancer and Leukemia Group B (CALGB) 90006 trial of bevasizumab incombination of docetaxel, and estramustine with a premedication with decadron inchemotherapy-naıve CRPC delivered promise (with the majority of patients achieving450% PSA reduction)314 and led to another two new clinical trials. The ongoing clinicaltrials are an National Cancer Institute—phase-II study of a four-drug combination strategyof docetaxel, prednisone, thalidomide, and bevacizumab in men with chemotherapy-naıveprogressive CRPC, and a CALGB phase III, double-blind, placebo-controlled trial of doc-etaxel plus prednisone with or without bevacizumab.

B. Small Molecule Inhibitors for VEGFR Tyrosine Kinase Activity

1. SU5416 (semaxanib)SU5416 is one of small molecule inhibitors of VEFR-associated tyrosine kinaseactivity.315,316 Huss et al. demonstrated when SU5416 was administered to TRAMP micebetween 16 and 22 weeks of age, therapy directed specifically against the VEGFR signalingaxis can dramatically impair angiogenesis and induce apoptosis of autochthonous sponta-neous and progressive CaP.316 Further preclinical studies using PC-3 human CaP xenografts,demonstrated that a combination regime of the VEGFR-2 inhibitor, SU5416 with anotherpotent anti-angiogenesis agent, endostatin, led to a significant delay in the onset of tumorprogression.317 In a randomized phase II trial, no disease modifying effects of SU5416 weredetectable in this small study (36 CaP patients). Modest toxicity, an inconvenient adminis-tration schedule, and availability of other VEGFR-targeted agents support the decision tohalt further evaluation of SU5416 in CaP treatment.318

2. AZD2171AZD2171 is a potent, selective, ATP-competitive small molecule that inhibits all VEGFreceptors.319 Wedge et al. demonstrated in vitro and in vivo inhibition of VEGF-stimulatedproliferation in cancer.320 A phase I dose-finding study using AZD2171 was conducted in 83patients with advanced solid tumors.321 Of the 83 patients enrolled, only 1 patient had CaP,and this patient was 1 of the 2 patients who had a confirmed partial response. Adverse eventsincluded fatigue, diarrhea, nausea, dysphonia, hypertension, anorexia, and vomiting. Themost common dose-limiting toxicity was hypertension (7 patients) and 2 patients presentedwith hypertensive crisis, all occurring at doses of 20mg. Ryan et al. have reported a phase Istudy exploring the safety and pharmacokinetics of AZD2171 in CaP.322

3. SorafenibOne of the pathways of angiogenic inhibition is through the use of the novel biaryl ureacompound sorafenib. It functions as a multityrosine kinase inhibitor and has been shown inpreclinical models to inhibit wild-type and mutant b-Raf and c-Raf kinase isoforms invitro.323 In addition, this agent also inhibits p38, c-kit, VEGFR-2, and platelet-derivedgrowth factor receptor-b (PDGFR-b), affecting tumor growth as well as possibly promotingapoptosis by events downstream of c-Raf.324,325 Not only does it exhibit tumor inhibition in



Ras mutated genes but it also shows blockade of tumor angiogenesis through inhibition ofVEGFR-2, VEGFR-3, and/or PDGFR-b. A phase II study of an initial 22 patients usingsorafenib in metastatic CRPC was presented in 2006.326 Although no patients showed PSAdeclines 450%, there was discordance between a rise in PSA and improvement in bonylesions by bone scintigraphic scans in two patients. This led to further accrual in the trial to atotal of 46 patients. Another phase II trial presented in the same year showed that of anevaluable 42 patients, 15 (35.7%) showed stable disease of Z12 weeks and 25 (59.5%) hadprogressive disease.327 Currently a phase II trial combining sorafenib with either docetaxeland prednisone or mitoxantrone and prednisone in patients with CRPC who haveprogressive disease despite systemic chemotherapy is accruing patients (NCT00414388200751).

C. Targeting Platelet-Derived Growth Factor Alpha (PDGF-a)

1. ThalidomidePreclinical experiments revealed that treatment with N-substituted thalidomide, a potentangiogenesis inhibitor, analog CPS11, (targeting PDGF-a) led to 90% inhibition of tumorgrowth and 64% reduction in tumor vascularity of PC-3 human prostate xenografts.328

Angiogenesis inhibition in in vitro and in vivo animal models has been demonstrated bythalidomide, with pro-apoptotic features or reduction in angiogenesis factors, such as VEGFand bFGF, in patients with CaP.329

An open trial of the efficacy and safety of thalidomide in 20 patients with AI CaPresulted in significant reduction in PSA levels in 35% of patients.330 CaP patients with CRPCexhibited a relatively good response to a combination treatment of docetaxel and thalido-mide, by achieving a significantly improved median overall survival (28.9 months) comparedwith docetaxel-alone (14.7 months).331 In a triple-combination strategy of paclitaxel anddoxorubicin with thalidomide, 9 of 12 patients showed a 50% decrease in PSA.332 Combi-national approaches using granular-macrophage colony stimulating factor (GM-CSF) havebeen reported with some therapeutic promise. GM-CSF regulates the dendritic cell andtumor-specific cytokine T-cell-mediated response,333 and clinically, GM-CSF in combinationwith thalidomide results in a significant PSA response (23%).334

An open-label phase II randomized trial comparing low-dose (200mg/d) and high-dose(up to 1200mg/d) thalidomide was conducted in 63 patients (50 patients in the low-dose arm,and 13 patients in the high-dose arm) and reported.335 The high-dose arm was not welltolerated owing to the known side effects of sedation and fatigue, and no significant declinesin PSA were observed. A total of 28% of all patients had a 440% decline in PSA, and 18%of patients in the low-dose arm had a 450% PSA decline. In another randomized phase IIstudy docetaxel was added to thalidomide for the treatment of metastatic CRPC.336 In thistrial, 75 patients with chemotherapy-naıve metastatic CRPC were randomly assigned toreceive either docetaxel at 30mg/m2 intravenously weekly for 3 consecutive weeks, followedby a 1-week rest period (n5 25), or docetaxel at the same dose and schedule, plus thalido-mide 200mg orally each day (n5 50); this study was designed before the results of theTAX327 trial, which showed superiority of the every 3-week regimen of docetaxel.337 PSAconsensus and Response Evaluation in Solid Tumors criteria were used to determine theproportion of patients with a PSA decline and time to progression. Results showed that theproportion of patients with a 450% decline in PSA was higher in the docetaxel/thalidomidegroup (53% in the combined group vs. 37% in docetaxel-alone arm), after a median followupof 26.4 months. The combined docetaxel/thalidomide arm was superior in terms of medianprogression-free survival (5.9 months vs. 3.7 months in the docetaxel-alone arm). Updatedanalysis after a median followup of 47 months showed a statistically significant difference in

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median overall survival for the combined group (25.9 months) vs. the docetaxel-alone arm(14.7 months) (P25 0.0407).338 The regimen was fairly well tolerated, except for the initialobservation of thromboembolic events in the combination. Twelve of the first 43 patients inthe combined group developed thromboembolic events. These events prompted the routineprophylactic use of low-molecular weight heparins for prevention of thromboembolism, andno further events were noted.

These encouraging results led to further clinical trials investigating the use of thalidomideand docetaxel with other agents. Thalidomide was added to docetaxel and estramustine and18 of 20 patients had a PSA decline of 450% with 2 of 10 patients with soft-tissuelesions achieving a partial response.339 However, the toxicity of estramustine precludes thiscombination from further study.

D. Endogenous Angiogenesis Inhibitors

A large number of endogenous anti-angiogenic factors have been identified, includingangiostatin, endostatin, and thrombospondin-1 (TSP-1).340 Angiostatin is a 38 kDa fragmentof plasminogen that can be secreted by a primary tumor and suppresses the growth ofmetastases in experimental animal models.341 Endostatin, a 20 kDa fragment of collagenXVIII and the most extensively studied endogenous angiogenesis inhibitor, regulates avariety of pro-angiogenic and anti-angiogenic factors342 and a large downstream signalingnetwork.343 TSP-1 is a disulfide-linked homotrimeric adhesive glycoprotein that mediatescell–cell and cell–matrix interactions.344 It is a major component of platelet a-granules, andits release from activated platelets significantly increases plasma TSP-1levels.345 TSP-1 is alarge ECM protein of 450 kDa that inhibits endothelial cell proliferation and capillarynetwork formation by binding to endothelial cell matrix proteins.346 TSP-1, by binding toendothelial cell surface receptor CD36, induces endothelial cell apoptosis through a p59fyn,caspase 3, and p38 MAPK signaling pathway.347

In several murine tumor models, systemic administration of angiostatin resulted indormancy of metastases.348 Galaup et al. developed a combined therapy to target both theepithelial and endothelial components of CaP.349 In vivo, the cytotoxic effect of angiostatinwas enhanced by the administration of docetaxel and total regression was observed in 83% ofPC-3 nude mouse xenograft tumors receiving the combination therapy.349 Angiostatindirectly inhibits human CaP cell invasion by blocking plasminogen binding to its cellularreceptor, CD26.350

Systemic administration of Endostatin showed potent inhibition of angiogenesis,maintenance of metastases at microscopic size, and regression of primary tumors.351

Li et al.’s recent study indicated that a replication-deficient adenovirus expressing endostatinand angiostatin fusion gene (EndoAngio) and a prostate-restricted, replication competentadenovirus (PRRA) showed dramatic antitumor efficacy.352 EndoAngio–PRRA uniquelycombines three distinct antitumor effects to eliminate AI CaP: anti-angiogenesis, viraloncolysis, and apoptosis.352 This novel anti-angiogenic PRRA represents a powerful agentfeasible for future clinical trials for CaP therapy.

The intratumoral administration of a TSP-1 expression vector into AI DU-145 CaPxenografts inhibits tumor growth in vivo through inhibition of tumor angiogenesis.353

In human CaP tissues, results have been somewhat equivocal. Although some studies havereported a correlation between TSP-1 and anti-angiogenesis,354,355 and between increasedTSP-1 expression in primary tumors and androgen deprivation in patients with CaP,356

others have found no such association.357,358 Using antibody array profiling, Shafer et al.reported serum TSP-1 could be a marker to distinguish BPH from CaP, which could leadto a reduction in the number of unnecessary prostatic biopsies.359 Endogenous angiogenesis



inhibitors are promising to treat CRPC for combination therapy with chemotherapy in thefuture.

E. Others

1. DoxazosinExperimental evidence indicates that the quinazoline-based compounds, such as doxazosinand terazosin (clinically used for treatment of hypertension and BPH), may also function ininducing prostate smooth muscle cell death via apoptotic pathways involving activation ofTGF-b1-mediated apoptotic signaling and inhibition of Akt.360 Growing evidence alsoimplicates anoikis (ECM detachment-induced apoptosis) in CaP epithelial and endothelialcells in response to quinazolines,361 potentially via targeting VEGF-mediated angiogenicresponse.361

2. SuraminSuramin is a polysulfonated naphtylurea previously used for the treatment of patients withtrypanosomiasis, onchocerciasis, and AIDS. It has the ability to bind several growth factorsand can antagonize their growth-promoting effects. Furthermore, suramin has demonstrateddirect antiproliferative effects against CaP cell lines.362,363

The therapeutic efficacy of suramin was analyzed in a large population of patients (390)with advanced CaP with a randomized dosing. The objective response rate was 9–15%, whilethe PSA response rates were 24–34%.364 In a randomized phase-III trial of suramin incombination with hydrocortisone conducted in 460 CRPC patients, patients receivingsuramin had higher PSA response rate, compared with the hydrocortisone arm,365 but still,serious limitations are associated with the clinical efficacy of this agent.

7. CONCLUSIONS AND FUTURE DIRECTIONS

Angiogenesis plays a critical role in tumorigenesis and metastasis in CaP. The mechanismsunderlying these changes are not fully elucidated yet. Angiogenesis is one of many discretesteps in tumor progression. Angiogenesis itself is a complex, multistep process that followsstage- and tissue-specific regulations. Although much effort has been put into the identifi-cation of the still-growing number of endogenous angiogenic inhibitors and activators, muchless is known about how these factors work together to form vessels at any given stage oftumor development. For example, what does it mean if a tumor expresses a certain number ofangiogenesis activators and inhibitors, with regard to its growth requirements? How do thesefactors interact to form tumor vessels, and how does this differ from the formation of anormal blood network? What happens when tumors co-opt blood vessels? Do those vesselsundergo changes and have signs of activated endothelium that would respond to ananti-angiogenic therapy? These are only a few questions that need to be addressed to have abetter understanding of the angiogenic switch.

Despite the recent advances in the management of patients with CRPC, there is muchroom for improvement. Anti-angiogenic therapy has much potential as stand-alone therapyfor transforming CaP into a chronic disease or in combination with the available therapies, toprovide curative approaches with decreased treatment-related morbidities. Furtherunderstanding of the angiogenic cascade will allow for additional molecular targets, whileadvancements in molecular delivery approaches, such as gene therapy or targeted therapy,will allow for the development of safe and effective treatment strategies. Anti-angiogenicagents can be used not only for the treatment of cancer, but also for the prevention of cancerrecurrence or metastasis. Given the heterogeneity of tumor and blood vessel growth,

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a multidrug approach that targets various factors might be more successful than mono-therapy in restraining cancer growth. The combination of anti-invasive and antimetastaticdrugs with anti-angiogenic drugs would seem to have particular promise. Another promisingavenue involves multitargeting of parallel pro-angiogenic signaling circuits, aimed to cir-cumvent preexisting redundancy or evasive mechanisms involving upregulation of alternativeangiogenic signaling circuits. Tumors at this stage have already activated various pathwaysthat allow them to easily override the angiogenic restrictions of one inhibitor. The mostsuccessful approaches are likely to involve combinatorial strategies to target cancer cellsthemselves, along with the stroma (that is, endothelial, perivascular, and inflammatory cells).

In CaP, as in other cancers, the single anti-angiognenic agent activity has been low, butthe addition of these agents to chemotherapy and gene therapy may be the area in which theyprovide their greatest clinical benefit. Other nonspecific treatments that include angiogenesissuch as genistein and histone deacetylase (HDAC) inhibitors, cytokine pathways, andhyperthermia should be considered in future CaP therapy. It is worthwhile to investigategenes that are involved in the stress response (i.e. hypoxia) as targets for CaP treatment.Future studies may also test the efficacy of anti-angiogenic strategies in earlier-stage disease,the use of oral agents targeting the VEGF receptor tyrosine kinase, and further combinationsof anti-VEGF agents and systemic therapies. The challenge is to identify robust biomarkersfor tumor angiogenesis. Indeed, biomarkers could become essential to justify the cost oftargeted therapies, by increasing the likelihood of benefit to a level that is acceptable topatients and clinicians.

8. ABBREVIATIONS

ADT androgen deprivation therapyAI androgen-independentAng angiopoetinAR angrogen receptorBPH benign prostate hyperplasiaBMDCs bone marrow-derived cellsCALGB cancer and Leukemia Group BCaP prostate cancerCRPC castrate-resistant prostate cancerDHT dihydrotestosteroneECM extracellular matrixEMMPRIN extracellular matrix metalloproteinase inducer proteinEMT epithelial–mesenchymal transitionERK extracellular signal-regulated kinaseET endothelinETAR endothelin A receptorETBR endothelin B receptorFDA federal drug administrationFGF2 basic fibroblast growth factorGCPII glutamate carboxypeptidase IIGM-CSF granular-macrophage colony stimulating factorHAMA human antimouse antibodyHDAC histone deacetylaseHIF hypoxic-inducible factorHGF hepatocyte growth factor



HGPIN high-grade intraepithelial neoplasiaIGF-1 insulin-like growth factor-1IGFBP insulin-like growth factor binding proteinIL8 interleukin 8LRP lipoprotein receptor-related proteinMAb monoclonal antibodyMAP mitogen-activated proteinMAPK mitogen-activated protein kinaseMT1-MMP membrane type1-MMPMMP matrix metalloproteinaseMVD microvessel densityPRRA prostate-restricted, replication competent adenovirusPI3K PI3 kinasePIN prostatic intraepithelial neoplasiaPD-ECGF platelet-derived endothelial cell growth factorPDGFR-b platelet-derived growth factor receptor-bPDGF platelet-derived growth factorPSA prostate-specific antigenPSMA prostate-specific membrane antigenRP radical prostatectomyTGF-b transforming growth factor-betaTie-2 tie-2 receptorTI-MMP tissue inhibitors of matrix metalloproteinaseTNFa tumor necrosis factor aTRAMP transgenic adenocarcinoma of the mouse prostateTSP-1 thrombospondin-1uPA urokinase plasminogen activatorVEGF vascular endothelial growth factorVEGFR vascular endothelial growth factor receptor

ACKNOWLEDGMENTS

We thank the Australian Research Council (ARC) and Cancer Institute NSW for fundingsupport. Dr. Yong Li is a Cancer Institute NSW Research Fellow.

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Yong Li completed his Ph.D. degree in Faculty of Medicine in 2000 from the UNSW, Sydney,Australia, and postdoctoral training at Cancer Care Center, St George Hospital, Sydney,UNSW, Australia, and then became an independent investigator. Currently, Dr. Li is a SeniorCancer Institute NSW Research Fellow and the Head of Cancer ResearchProgram, St George Hospital and Conjoint Senior Lecturer, Faculty of Medicine at UNSW.Dr. Li’ research program is aimed at (a): To investigate novel biomarkers or tumor-associated antigens for cancer diagnosis, monitoring and therapy; (b): To use targeted cancertherapy and combination therapy to control micrometastatic prostate cancer and ovarian can-cer; (c): To investigate the mechanism of cancer metastasis. These studies will provide newinsights into the mechanisms of cancer metastasis, control of micrometastatic development,which may lead to the development of novel therapies for the treatment of cancerprogression. The success in identifying proteins of interest in cancer patients may be used tostage the disease, monitor cancer progression and recurrence, and to guide clinicians in choosingthe best treatment methods for an individual patient.

Paul Cozzi is a Senior Urologist at the Department of Surgery, Urology Sydney and SeniorLecturer at UNSW. He has pioneered new surgical treatments for prostate cancer in theAustralian setting and established a database of patients with urological cancers,including prostate cancer patients at St George Hospital. His interests at present include clinicaland laboratory research into prostate and bladder cancer with a special interest in experimentaltherapeutics for the treatment of these malignancies and early diagnosis of cancer.

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Angiogenesis as a strategic target for prostate cancer therapy