Anti-angiogenesis therapy in cancer: Current challenges and future perspectives

Cancer Letters 320 (2012) 130–137

Contents lists available at SciVerse ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Mini-review

Anti-angiogenesis therapy in cancer: Current challenges and future perspectives Farbod Shojaei ⇑ Oncology Research Unit, Pfizer La Jolla, CA 92121, United States

a r t i c l e

i n f o

Article history: Received 27 January 2012 Received in revised form 4 March 2012 Accepted 5 March 2012

Keywords: Tumor angiogenesis VEGF Anti-angiogenesis therapy Drug resistance

a b s t r a c t It has been nearly 9 years since the FDA (Food and Drug Administration) approved the first antiangiogenic drug (bevacizumab) for treatment of metastatic colorectal cancer. Other angiogenic inhibitors have since been approved or are in different stages of clinical trials. However, continued clinical and preclinical investigations have identified major drawbacks associated with the application of this class of agents, including inherent/acquired resistance and induction of tumor invasiveness. In addition, lack of thoroughly validated predictive biomarkers has been one of the major hurdles to stratify cancer patients and to monitor tumor progression and response to the therapy. Investigations in clinic and preclinical models have provided some molecular and cellular mechanisms for the above challenges. This review aims to provide a concise update from recent findings. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 1.1. Angiogenesis in health and disease In embryonic life, formation and growth of new blood vessels from pre-existing vasculature is a critical process leading to formation of stable vasculature comprised of endothelial cells (ECs), mural cells (pericytes) and basement membrane in the adult [1].

In normal vasculature ECs are the innermost layer of vascular walls and are in direct contact with pericytes along the length of the vessels whereas the basement membrane is a uniform and thin layer that covers almost the entire length of ECs [2]. Vasculature in healthy adult is very stable and with the exception of rare events such as cyclical growth of vessels in the ovarian corpus luteum or during pregnancy, angiogenesis activities are rare in adult individuals [3].

In addition to normal development, angiogenesis is known to be an important event in pathological conditions such as tissue repair during wound healing and in tumor growth [4]. However, components of tumor vasculature and structures are distinct compared to normal vessels [5]: (i) tumor vessels lack the hierarchy of arterioles, capillaries and venules; (ii) tumor vasculature is disorganized and tortuous; (iii) vessels in tumors are leakier than their counterpart in normal tissues since TAECs (tumor associated ECs) are not in close contact with pericytes and are loosely connected to basement membrane; (iv) in some circumstances tumor cells may line blood vessels via vasculogenic mimicry; and (v) compared to ECs ⇑ Address: Pfizer Global R&D, 10724 Science Center Dr., San Diego, CA 92121, United States.

E-mail addresses: Farbod.shojaei@pfizer.com, farbodshojaei@hotmail.com 0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2012.03.008

isolated from non-tumor tissues, several transcripts including PlGF, CD137, CD276 and CD109 have been found to be enriched in TAECs indicative of their distinct molecular properties.

Despite extensive studies into the cellular and molecular properties of TAECs, the origin of these cells in tumors is still heavily debated [6]. For example, while a considerable amount of data in certain models identifies EPCs (endothelial progenitor cells) as one of the subsets of BMMNCs (bone marrow mono-nuclear cells), directly contribute to tumor vasculature, series of other studies suggest that contribution of BM-EPC in the vasculature is modeldependent, transient and limited to early angiogenesis events in the tumors [7].

Irrespective of cellular origin, induction of angiogenesis requires a shift/switch towards activation/upregulation of inducers of angiogenesis over suppression of angiogenic inhibitors (hereafter AI). Some key angiogenic activators include VEGF-A (vascular endothelial growth factor A hereafter VEGF) [8], MMPs (matrix metalloproteinases), PlGF (placenta growth factor), FGF (fibroblast growth factor) and HGF (hepatocyte growth factor) [9]. Endogenous inhibitors of angiogenesis include thrombospondins (THSBs) endostatin, angiostatin and cytokines such as interleukin-12 [10].

Overall, role of angiogenesis in homeostasis in normal tissues and in tumor growth and expansion is very well established.

1.2. VEGF inhibitors are key component of anti-angiogenesis therapy Given the role of angiogenesis in tumor growth and progression, targeting tumor vasculature as a therapeutic mean has been long proposed and inhibition of growth factors/signaling pathways necessary for ECs growth and proliferation is one of the practical approaches to inhibit tumor angiogenesis [4,11]. Among angiogenic

131

F. Shojaei / Cancer Letters 320 (2012) 130–137

activators, VEGF was proven to be one of the key regulators of both physiological and pathological angiogenesis [12]. At the genetic level, loss of a single VEGF allele is embryonically lethal because of major defects in vascular system [13]. Moreover, VEGF-null embryonic stem cells significantly lost their ability to form teratoma in the recipient after injection in testis capsule demonstrating a key role for angiogenesis (and consequently VEGF) in tumor growth.

HIF-1a (hypoxia inducible factor), among many factors [14], is one of the key regulators of VEGF expression since it can bind to VEGF promoter [15]. Rapid proliferation of tumor cells and poor blood flow suggest a hypoxia-conducive environment in different areas of tumors resulting in rapid upregulation of VEGF [16]. Consistently, VEGF is highly enriched in a variety of human tumors [17] while VEGFR1, VEGFR2 and VEGFR3 are highly expressed in TAECs [18].

To further support role of VEGF in tumor angiogenesis and from a therapeutic standpoint, treatment of mice carrying human tumors with an anti-VEGF neutralizing Mab (monoclonal antibody) significantly inhibited tumor growth in the recipients [19]. In 2003, the FDA (Food & Drug Administration) approved Bevacizumab (AvastinÒ; Genentech Inc.), a humanized variant of a VEGF neutralizing Mab, [20] as the first anti-angiogenic agent for combinatorial treatment with SOC (standard of care) in metastatic CRC (colorectal cancer) [21] and subsequently for treatment of patients suffering from NSCLC (non-small-cell lung cancer) [22] or MBC (metastatic breast cancer) [23]. Since then, several VEGF inhibitors targeting VEGF or its receptors, are in different stages of clinical development. For example VEGF-TrapR1R2 (Aflibercept; Regeneron Inc.), a chimeric soluble receptor containing structural elements from VEGFR1 and VEGFR2 [24], has the ability to bind to and neutralize circulating VEGF. VEGF-TrapR1R2 has shown superior anti-tumor activity compared to other VEGFR-blockers (i.e.

DC101; ImClone System) in preclinical models and is currently in clinical trials. Additionally, inhibition of VEGF pathway through blockade of VEGF receptors inhibits tumor growth [25,26]. For example small molecule RTKIs (receptor tyrosine kinases inhibitors) targeting VEGF and other signaling pathways have recently been developed. Some of the most clinically relevant RTKIs include sunitinib (SU11248; Sugen) [27], pazopanib (Votrient; GSK) [28], sorafenib (Bay 43-9006; NexavarÒ) [29] vendatanib (Caprelsa; AstraZeneca) [30], cabozantinib (XL184; Exelixis) [31] and most recently axitinib [32], tivozatinib [33] and linifanib [34,35]. Table 1 has summarized some of the characteristics of these agents including their main target and indications in cancer patients.

Despite all the clinical indications, detail of mechanism of action of anti-angiogenic compounds particularly in cancer patients remains to be unraveled. Given a key role for VEGF in angiogenesis, series of studies in preclinical models suggest that AIs suppress tumor growth via inhibition of tumor angiogenesis. However, investigations pioneered by Rakesh Jain in recent years indicate that anti-angiogenics may act through vascular normalization which

implies that AIs target the non-functional and redundant vessels in tumors resulting in reduction in interstitial fluids, improve tumor oxygenation and, when combined with SOC, enhance delivery of cytotoxic agents to the tumor mass [36].

It is noteworthy to mention that while almost all VEGF-inhibitors (antibodies and small molecule inhibitors) block angiogenesis and tumor progression in preclinical models, clinical trials have brought different outcomes when comparing antibodies to small molecule inhibitors. For example Bevacizumab in combination with SOC has been successfully approved for CRC [21], NSCLC [22] and MBC [23]. However, sunitinib trials failed to provide the same outcome in the above cancers possibly due to complexity of human trials and differences in the kinetics of antibodies vs.

RTKIs [37]. Together, identification of VEGF and development of angiogenic inhibitors provided new therapeutic avenues for cancer patients.

It is necessary to mention that in addition to VEGF inhibitors, VDAs (vascular disrupting agents) are a completely different class of anti-angiogenics and suppress tumor growth through induction of vascular collapse causing hypoxia and necrosis in tumor mass [38]. ASA404, a flavonoid compound, is one of VDAs capable of induction of apoptosis in TAECs resulting in the inhibition blood flow in tumor mass. ASA404 is currently in advance stage of clinical development in combination with SOC in NSCLC patients [39].

2. Challenges associated with anti-angiogenic therapy Despite the initially promising performance of AIs in the clinic, anti-angiogenesis therapy is facing three major challenges including inherent/acquired resistance, enhanced invasiveness during treatment with AIs and lack of validated predictive biomarkers to select patient population and to monitor tumors responses to the therapy. Fig. 1 illustrates these challenges in anti-angiogenesis therapy (Fig. 1).

2.1. Resistance to AIs 2.1.1. General characteristics and role of tumor cells Most cancer patients eventually demonstrate lack of response to anti-angiogenesis therapy while on treatment regimen. Molecular and cellular mechanisms mediating resistance have been extensively studied [40]. Some characteristics of inherent/acquired resistance to AIs include: (i) both tumor and non-tumor compartments contribute to resistance to anti-angiogenics; (ii) resistance to AIs may occur independent of class of agents i.e. antibodies [41] or small molecule inhibitors [42], (iii) resistance occurs independent of affinity of the anti-VEGF antibody [43]; (iv) limited bioavailability of therapeutic agent in tumor mass might account for lack of response to the therapy [44]; (v) other angiogenic factors such as NRP1 (Neuropilin1) which enhances VEGF binding to the receptor or activate ECs migration and adhesion may mediate

Table 1 VEGF RTKIs and their indications in cancer patients.

Compound

Company

Target(s)

Indications

References

Sunitinib (SU11248) Pazopanib (Votrient) Sorafenib (Bay 43-9006; NexavarÒ) Vendatanib (Caprelsa)

Pfizer GSK Bayer

VEGFRs, PDGFRB, CSF1R, c-Kit VEGFRs, PDGFRs and c-kit VEGFR-2 and -3, PDGFR-b, Flt-3, c-kit, Raf kinases VEGFRs and EGFRs, RET-tyrosine kinases

RCC, GIST RCC, RCC, Inoperable HCC

[27,113,114] [28] [29,115,116,117] [30,118]

Cabozantinib (XL184) Tivozanib (AV-951, KRN-951)

Exelixis AVEO Pharmaceutical Pfizer Abbott

VEGFRs Met VEGFRs

Late-stage Medullary Thyroid Cancer Prostate Cancer RCC

VEGFRs VEGFRs, PDGFRB, CSF1R,

mRCC RCC, NSCLC

[32] [34,35]

Axitinib (AG-013736) Linifanib (ABT-869)

AstraZeneca

[31,119] [33]

132

F. Shojaei / Cancer Letters 320 (2012) 130–137

Fig. 1. Resistance, acceleration of metastasis and lack of predictive biomarkers of response present major challenges in the field of anti-angiogenesis therapy. In the absences of predictive biomarkers, tumors treatment with AIs may have three outcomes in preclinical models and/or in cancer patients. The first outcome is full response in established tumors to AI treatment where tumor growth and angiogenesis is significantly suppressed. Sensitive tumors have been identified in preclinical models but have yet to be fully diagnosed in the clinic. The second scenario is acquired resistance and occurs when tumors respond to AIs at the beginning of treatment but become gradually resistant to the therapy. The third outcome is inherent resistance or refractoriness and covers tumors that never respond to AIs. In both acquired resistant and refractory tumors, alternative angiogenic factors such as FGF2, Bv8, G-CSF, PlGF, PDGFC and HGF play important roles in mediating resistance to AIs. These factors may be released by cancer cells, stromal cells or both cell types in tumor mass. Irrespective of mode of response, treatment with AIs may also accelerate metastasis to other organs, however, whether response of primary tumors play any role in increased-invasiveness remains to be determined. Resistance to AIs and enhanced metastasis further signify identification of biomarkers to select patient populations that are more responsive to AIs and may not develop invasive lesions as a result of treatment with these agents.

resistance to AIs [45]; (vi) incomplete abolishment of basement membrane and pericytes is another reason for lack of response to AIs [46].

Tumors may serve as a source of alternative angiogenic factors when VEGF is inhibited. For example treatment of RIP-Tag tumors (rat insulin promoter T antigen) with a VEGFR2 inhibitor resulted in upregulation of FGF2 in the tumors leading to lack of response to the treatment [47]. Cancer stem cells (or tumor initiating cells) may also mediate resistance to AIs [48–50]. Investigations by Wang et al. showed that a subset of CD133 + glioblastoma cancer stem cells have the capacity of differentiation to vascular endothelium in a Notch driven manner and independent of VEGF [50]. ECs express notch receptors and are a source of DLL4 (Delta-like 4 ligand) that can activate the pathway through an autocrine loop [51]. Blockade of DLL4 inhibited tumor growth in several xenograft models [52,53] and showed additive efficacy when combined with anti-VEGF Mab. Histology analyses showed that anti-DLL4 increased proliferation of TAECs but inhibited their differentiation to functional vessels [53].

From translational research standpoint, kinetics of tumor growth and angiogenesis in preclinical models especially subcutaneous tumors may not be representative of the same mechanisms in cancer patients and therefore preclinical findings may not be fully translatable to the clinic. Furthermore, in most of clinical indications AIs are only efficacious when combined with SOC and resistance to the therapy may originate from AIs, SOC or combination of both compounds. Therefore, it is extremely challenging to dissect mechanisms of resistance to anti-angiogenics in combination treatment settings.

2.1.2. Stromal cells and resistance to AIs Stromal compartment (heterogeneous population of cells including infiltrating blood cells, fibroblasts, pericytes and mesenchymal cells) plays a significant role in mediating resistance to AIs. For example studies in mouse models of human lung adenocar-

cinoma showed upregulation of EGFR and FGFR in stroma but not tumor cells [54]. Combination of Bevacizumab and an EGFR inhibitor circumvented resistance to anti-VEGF inhibition and increased progression free survival. Stromal cells employ the following mechanisms to mediate resistance to AIs: (i) direct contribution into tumor vasculature; (ii) release of angiogenic factor and (iii) averting immune surveillance from tumor cells [55].

Myeloid cells, identified as CD11b + Gr1+, are one of the major components of stroma and have been extensively studied for their role in tumor growth and in mediating resistance to anti-angiogenics [55]. Myeloid cells are recruited by resistant tumors and contribute in VEGF-independent angiogenesis via release of angiogenic factors such as Bv8 [41]. Further studies identified G-CSF (granulocyte colony stimulating factor) as a key regulator of Bv8 expression and showed that inhibition of G-CSF can also suppress tumor growth as single agent or in combination with anti-VEGF antibody to similar extent that was observed with anti-Bv8 treatment [56]. Inhibition of G-CSF or Bv8 also significantly reduced lung metastasis indicating a dual role for these molecules in tumor angiogenesis and invasiveness [57]. Recent study also showed that BMMNCs are an enrich source of HGF (hepatocytes growth factor) that can trigger tumor growth and angiogenesis in sunitinib-resistant tumors [42]. Myeloid cells may affect bioavailability of VEGF for its receptors in the tumors via release of MMP9 [58]. Finally, myeloid cells promote tumor growth via suppression of immune response since they are a rich source of PGE2 (prostaglandin E2), ARG1 (arginase-1) and NOS2 (nitric oxide synthase) and also induce development of CD4 + CD25 + Foxp3 + T regulatory cells that are anergic and immunosuppressive [59].

In addition to myeloid cells other components of stroma also contribute in VEGF-independent tumor growth. TAFs (tumor associated fibroblasts) are a source of cytokines and growth factors such as VEGF, CXCL12 and PDGFC (platelet derived growth factor) [60]. Finally, pericytes are in charge of maintaining vascular stability via matrix deposition and also through release of angiogenic

F. Shojaei / Cancer Letters 320 (2012) 130–137

factors such as angiopoietin-1 (Ang-1) which is a ligand for the Tie2 receptor [61]. Overall, these studies indicate an important role for stroma in mediating tumor growth and resistance to antiangiogenics. Of note, Stroma and vasculature are only representing a minor subset of cells in tumor mass making it a challenging task to understand mechanisms underlying resistance and/or metastasis. In addition, unlike cancer cells that carry genetic aberrations, cells of stroma are genetically stable but are permanently influenced or instructed by tumor cells in their microenvironment.

Therefore, molecular and cellular properties of stroma may drastically change in different tumor types and more specifically in resistant vs. sensitive tumors suggesting that stroma and vascular characteristics are rather tumor dependent. Overall, resistance to AIs is a multifactorial complex process and is centralized by cancer cells.

2.2. Acceleration of metastasis by anti-angiogenic compounds Recent reports in preclinical models indicated that antiangiogenic agents may enhance tumor invasiveness and metastasis. Investigations by Pa¯ez-Ribes et al. showed that treatment of mouse models of glioblastoma and pancreatic neuroendocrine carcinoma with VEGF-inhibitors increased invasiveness in the primary tumors and enhanced metastasis to liver and lymph nodes [62]. Conditional knockout of VEGF in the same models confirmed pharmacological findings. Consistently, treatment of glioblastoma tumors with anti-VEGF antibody promoted metastasis and invasiveness [63]. In another independent report Ebos et al. showed that pre-treatment of mice with AIs prior to i.v. injection of tumor cells increased lung metastasis and decreased survival in the recipients [64]. In addition, short-term treatment with sunitinib prior to mammary fat pad implantation of human tumor cells significantly increased metastasis after tumor removal and resulted in lower survival rate. The major caveats of the above report is administration of sunitinib at doses that are significantly higher than the clinical dose. We recently investigated metastasis and invasiveness in several tumor models using suntinib at/near clinical dose [65]. Our data indicate that suntinib induced metastasis depends on tumor type and may be mediated by c-Met pathway activation. Hypoxia and inflammatory responses are among the most appealing reasons for acceleration of metastasis during treatment with AIs. Role of hypoxia in tumor progression and metastasis is well established [66] and anti-angiogenesis therapy may induce formation of a hypoxic microenvironment leading to selection of clone of tumor cells capable of survival and growth in a low oxygen environment.

Tumor treatment with AIs may release inflammatory mediators by the host cells resulting in formation of per-metastatic niches and a conducive microenvironment for seeding of cancer cells.

Small-molecule RTKIs might trigger a more acute inflammatory response compared to large molecules, therefore causing a profound metastatic effect [67]. Besides, in contrast to selective anti-VEGF antibodies, multi-targeted RTKIs such as sunitinib may also target pericytes via PDGFRs potentially resulting in increased possibility of metastasis [68]. Future studies will determine if, among approved clinical indications, treatment with AIs might have induced metastasis in cancer patients.

2.3. Lack of biomarkers to monitor response to anti-angiogenic therapy Given above challenges and modest responses observed to antiangiogenesis therapy in the clinic, it is imperative to identify a set of biomarkers to select populations of likely responders and/or to monitor disease progression and response over the course of treatment in the clinic [69]. While reliable biomarkers have yet to be identified, promising data are emerging from recent studies [70].

133

For example CD31 expression and PDGFRA staining in histology have been proposed as biomarkers of response in breast cancer patients treated with bevacizumab and SOC [71]. Recently CAIX (Carbonic anhydrase IX) was found to act as a predictor of response in mRCC patients treated with sorafenib. Strikingly CAIX was found to be expressed only in ccRCC tumors (clear cell RCC) but not normal kidney [72]. Additionally, recent report in recurrent glioblastoma patients treated with a pan-VEGF RTKI showed an increase in the levels of circulating SDF-1a and FGF-2 in a subset of patients who were unresponsive to the therapy [73].

2.3.1. CEPCs (circulating endothelial progenitor cells) It was primarily hypothesized that quantification of CEPCs that are a subset of CECs and are released from bone marrow to blood circulation might be a reliable approach to monitor response to AIs [74]. The number of CECs (1–20 cells/ml of blood in normal individuals) significantly increases during tumor progression [75]. While few trials have shown a correlation between reduction in CECs and response to anti-angiogenic treatment [76] metronomic therapy showed a significant increase in the number of CECs [46]. In addition, there are conflicting reports about reliable markers to identify and to isolate populations of CECs and CEPCs making it a challenging approach as a potential biomarker of response to AIs. 2.3.2. Analysis of cytokines and/or growth factors in serum Quantification of circulating VEGF is another candidate biomarker of response to AIs in clinic [77]. However, circulating VEGF may not provide an accurate value as improper handling of platelets may release VEGF into the circulation [78]. Additionally, antiVEGF antibodies form complexes with circulating VEGF that is still measured as total VEGF thereby providing a false representative of free (unbound) VEGF in plasma [79]. Despite all of these caveats, plasma levels of free-VEGF increase during treatment with antiVEGF antibodies or small molecule RTKIs and are still regarded as a potential predictive biomarker of response to the therapy [80,81]. It is important to mention that alterations in levels of circulating VEGF might serve as a pharmacodynamic marker and may not have any predictive value with respect to patients’ response to treatment with AIs. Additionally lack of any supporting evidence on correlation between circulating VEGF and overall survival or progression free survival in AI-treated patients may challenge this approach as a predictive biomarker of response to antiangiogenics. Besides VEGF, soluble VEGFR2 and VEGFR3 have been associated with patient response to bevacizumab (breast cancer; [82]) or sunitinib (mRCC [83]).

In addition to VEGF family, analysis of gene expression profiles in tumor samples isolated from rectal carcinoma patients before and after treatment with bevacizumab showed that anti-VEGF mAb upregulated SDF1a, CXCR4, and CXCL6, in tumor cells and also induced expression of NRP1 in TAMs (tumor-associated macrophages). On the contrary PlGF and Ang2 were down-regulated in tumor cells and Ang1 expression was decreased in both tumor cells and TAMs [84].

2.3.3. Hypertension Hypertension is another biomarker of response to AIs as blockade of VEGF results in a reduction in NO (nitric oxide) synthesis leading to vasoconstriction and increase in blood pressure [85].

Analysis of blood pressure in CRC and MBC patients treated with bevacizumab and SOC indicated that patients with grade 2–4 of hypertension showed a significantly better survival and response rate [86,87]. The major caveat of using hypertension as a biomarker is that other physiopathological stimuli can also increase blood pressure in patients and so relying solely on hypertension may not be the best approach to measure efficacy of AIs.

134

F. Shojaei / Cancer Letters 320 (2012) 130–137

2.3.4. Imaging modalities Imaging technologies have also been employed to track tumors responses to AIs. Among several imaging modalities, DCE-MRI (dynamic contrast-enhanced magnetic resonance imaging) appears to be a reliable method to measure response to the therapy via measurement of surface area of ECs and vascular permeability [88,89].

Recently, VEGF-PET tracers (positron emission tomography) using anti-VEGF labeled compounds such as 89Zr-ranibizumab showed promising data as a non-invasive biomarker to monitor tumor growth in xenograft models during and after treatment with sunitinib [90].

2.3.5. SNP (single nucleotide polymorphism) Finally SNP analyses in VEGFs and VEGFRs have identified several nucleotides correlative of response to treatment with bevacizumab and SOC. Schneider et al. showed that VEGF-2578AA in patients who showed grade 3–4 hypertension with metastatic breast cancer had a better median overall survival compared to VEGF-2578CA and VEGF-2578CC genotypes [87]. Very recent study in mCRC patients showed that a SNP in VEGFR1-319C/A was correlated with response to the therapy as patient with VEGFR1-319AA genotype showed a significantly better objective response and response rate compared to VEGFR1-319CC + CA [91]. Moreover, analysis of polymorphism in several genes (VEGF-A, VEGFR2, VEGFR3, PDGFR-a, ABCB1, ABCB2, IL8, CYP3A4 and CYP3A5) in clear-cell RCC patients treated with sunitinib showed that two VEGFR3 missense polymorphisms (rs307826 and rs307821) are inversely correlated with progression free survival [92]. In conclusion, while emerging reports have provided promising data about reliable biomarkers to monitor efficacy of VEGF inhibitors there are still key questions remain to be clarified.

3. Conclusions and future perspectives Inhibitors of angiogenesis have provided new treatment avenues for cancer patients and have significantly improved our understanding of role of angiogenesis in tumor growth and metastasis. Further success in the field of anti-angiogenesis therapy awaits overcoming above challenges and identifying new approaches to translate recent findings in preclinical models to the clinic.

One viable approach is to develop rationale-based combination strategies to overcome resistance in the clinic. As mentioned above preclinical studies have identified and characterized alternative angiogenic factors (such as Bv8, PlGF, Dll4, HGF, FGF2 and PDGFC) mediating resistance to AIs. Current and future studies are focused on validating these targets in AI-treated patients with recurred tumors to identify any potential for combination treatment. Of note, targeting VEGF is a hallmark of all the AIs as several studies in preclinical models have shown that any anti-angiogenesis combination treatment should contain a VEGF inhibitor otherwise tumors will not respond to the therapy [41,47,54,56,93,94]. Therefore VEGF inhibition is an important component of any anti-angiogenic combination strategy.

From drug discovery standpoint, miRNAs (MicroRNA) and siRNAs (short interfering RNA) are an interesting area of investigation [95]. For example, miR-132 found to be expressed in the tumor vasculature but not normal ECs [96]. Over expression of miR-132 induced angiogenesis activity in ECs in vitro while anti-miR-132 significantly inhibited tumor growth and angiogenesis via upregulation of p120RasGAP which is a negative regulator of Ras pathway.

Future studies will determine therapeutic value of siRNAs and miRNAs in VEGF resistant models and also in the clinic. Several siRNA targeting VEGF family or receptors such as bevasiranib (OPKO Health) and AGN211745 (Sirna-027; Allergan) [97] are in different

stages of clinical trials for treatment of wet form of neovascular age-related macular degeneration, a disease featured by vascular leakage and angiogenesis in the macular region (central retina) of an eye [98].

Another approach is to employ new technologies to improve efficacy and potency of current anti-angiogenic compounds in the clinic. For example, tracing radiolabeled antibodies in cancer patients prior to surgical removal of primary and metastatic tumors showed that average concentration in the tumors is only 0.015% of the injected antibodies [99]. Nanoparticles (NP, 50– 200 nm size) have recently gained traction since they have greater permeability, better penetrance and retention due to leaky vasculature in tumors, and can stay longer in the tumor mass due to lack of draining structure [100]. For example, conjugation of an antiFLK1 mAb to radioisotope 90yttrium via a NP (anti-FLK1-NP-Yttrium) provided a more potent compound that significantly inhibited growth of human tumors via targeting tumor vasculature in preclinical models compared to controls [101]. Conjugation of cytotoxic agents to NP may also enhance tumor growth inhibition via targeting both vasculature and tumor cells. PEGylation (poly ethylene glycol) of cytotoxic agent increases their circulation time and decreases rapid clearance [102]. Formulation of oxaliplatin with PEG-coated cationic liposomes significantly inhibited tumor growth compared to non-formulated compound. In vivo imaging revealed a broader distribution and greater concentration of encapsulated oxaliplatin in the tumors. In addition, in vitro analysis also showed that the encapsulated oxaliplatin is internalized by both tumors and endothelial cells making this an attractive strategy to improve drug delivery and targeting both tumors and vasculature [103]. Targeting integrin binding site (RGD sequence arginine-glycine-aspartate) can also enhance drug delivery to the vasculature since TAECs are enrich in integrins (reviewed in [104]). Murphy et al. recently showed that encapsulating doxorubicin with RGD-NP resulted in a significant reduction in metastasis in pancreatic and renal cell orthotopic models without causing severe toxicity compared to free drug [105].

Finally targeted delivery of toxin agents to vasculature is another approach to improve potency of AIs. SLT (Shiga-like toxins) is one of the optimal toxin for this purpose since it directly binds to Gb3/CD77 expressed on ECs via its B subunit and can trigger cell death by inhibiting binding of eEF-1 (elongation factor)/aminoacyltRNA complex to ribosomes resulting in inhibition of protein synthesis [106]. SLT-VEGF121 (which lacks heparin binding domain) fusion significantly inhibited growth of VEGFR2-expressing porcine ECs in vitro [107] and inhibited tumor growth in several preclinical models via targeting VEGFR2 + ECs [108]. Similarly ADCs (antibody drug conjugates) are an attractive technology for the efficient and potent delivery of AIs (reviewed in [109]). In ADCs, an antibody that is conjugated to a toxin by a linker, serves as a vehicle to deliver toxin and/or cytotoxic agent selectively to tumor cells or ECs that express corresponding antigen on the cell surface.

Upon binding to the corresponding receptor, ADC is internalized and causes cell death by releasing the toxin. Therefore, factors such as differential expression of antigen/receptor on tumor or TAECs, stability of linker and efficacy of internalization play key roles in development of efficacious ADCs. Given the selective killing in ECs, any anti-angiogenic driven ADC should preferentially target TAECs but not healthy endothelial cells [110]. Therefore vascular targeting ADCs could also improve drug kinetics in tumors by selective targeting of TAECs. Among ADCs developed to target tumor vasculature, L19 (a mAb selectively targeting a specific splice form of fibronectin expressed on the vasculature) [111] and G11 and F16 (targeting C-terminal domain of tenascin-C that is highly expressed in brain and lung tumors and also in perivascular regions) [112] are in advance stages of clinical programs.

F. Shojaei / Cancer Letters 320 (2012) 130–137

In conclusion, while early achievements in anti-angiogenesis therapy have been associated with several challenges, recent advances in the areas of tumor angiogenesis and drug development may pave the path towards a more efficient and successful clinical application in the future.

[24]

[25]

Acknowledgements I thank Dr. Eric Fpm Schoenmakers for helpful comments and suggestions on this manuscript. I apologize to investigators whose reports were not cited in this manuscript due to space limitation.

References [1] J. Wilting, B. Christ, Embryonic angiogenesis: a review, Naturwissenschaften 83 (1996) 153–164, ISSN:0028-1042.

[2] J.A. Rhodin, H. Fujita, Capillary growth in the mesentery of normal young rats.

Intravital video and electron microscope analyses, Journal of Submicroscopic Cytology and Pathology 21 (1989) 1–34.

[3] W. Risau, I. Flamme, Vasculogenesis, Annual Review of Cell and Developmental Biology 11 (1995) 73–91.

[4] J. Folkman, Tumor angiogenesis: therapeutic implications, New England Journal of Medicine 285 (1971) 1182–1186.

[5] S. Hiratsuka, Vasculogenensis, angiogenesis and special features of tumor blood vessels, Frontiers in Bioscience 16 (2011) 1413–1427.

[6] D. Ribatti, The involvement of endothelial progenitor cells in tumor angiogenesis, Journal of Cellular and Molecular Medicine 8 (2004) 294–300.

[7] D. Gao, D. Nolan, K. McDonnell, L. Vahdat, R. Benezra, N. Altorki, V. Mittal, Bone marrow-derived endothelial progenitor cells contribute to the angiogenic switch in tumor growth and metastatic progression, Biochimica et Biophysica Acta 1796 (2009) 33–40.

[8] N. Ferrara, W.J. Henzel, Pituitary follicular cells secrete a novel heparinbinding growth factor specific for vascular endothelial cells, Biochemical and Biophysical Research Communications 161 (1989) 851–858.

[9] C.C. Khoury, F.N. Ziyadeh, Angiogenic factors, Contributions to Nephrology 170 (2011) 83–92.

[10] G. Taraboletti, M. Rusnati, L. Ragona, G. Colombo, Targeting tumor angiogenesis with TSP-1-based compounds: rational design of antiangiogenic mimetics of endogenous inhibitors, Oncotarget 1 (2010) 662–673.

[11] G.H. Algire, H.W. Chalkley, Vascular reactions of normal and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants, Journal of the National Cancer Institute 6 (1945) 73– 85.

[12] N. Ferrara, H.P. Gerber, J. LeCouter, The biology of VEGF and its receptors, Nature Medicine 9 (2003) 669–676.

[13] N. Ferrara, K. Carver-Moore, H. Chen, M. Dowd, L. Lu, K.S. O’Shea, L. PowellBraxton, K.J. Hillan, M.W. Moore, Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene, Nature 380 (1996) 439–442.

[14] O. Rosmorduc, C. Housset, Hypoxia: a link between fibrogenesis, angiogenesis, and carcinogenesis in liver disease, Seminars in Liver Disease 30 (2010) 258–270.

[15] G.L. Wang, G.L. Semenza, Purification and characterization of hypoxiainducible factor 1, Journal of Biological Chemistry 270 (1995) 1230–1237, ISSN:0021-9258.

[16] C. Brahimi-Horn, J. Pouyssegur, The role of the hypoxia-inducible factor in tumor metabolism growth and invasion, Bulletin du Cancer 93 (2006) E73– E80.

[17] L.M. Ellis, D.J. Hicklin, VEGF-targeted therapy: mechanisms of anti-tumour activity, Nature Reviews Cancer 8 (2008) 579–591.

[18] T.V. Petrova, P. Bono, W. Holnthoner, J. Chesnes, B. Pytowski, H. Sihto, P. Laakkonen, P. Heikkila, H. Joensuu, K. Alitalo, VEGFR-3 expression is restricted to blood and lymphatic vessels in solid tumors, Cancer Cell 13 (2008) 554– 556.

[19] K.J. Kim, B. Li, J. Winer, M. Armanini, N. Gillett, H.S. Phillips, N. Ferrara, Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo, Nature 362 (1993) 841–844.

[20] L.G. Presta, H. Chen, S.J. O’Connor, V. Chisholm, Y.G. Meng, L. Krummen, M. Winkler, N. Ferrara, Humanization of an anti-VEGF monoclonal antibody for the therapy of solid tumors and other disorders, Cancer Research 57 (1997) 4593–4599.

[21] H. Hurwitz, L. Fehrenbacher, W. Novotny, T. Cartwright, J. Hainsworth, W. Heim, J. Berlin, A. Baron, S. Griffing, E. Holmgren, N. Ferrara, G. Fyfe, B. Rogers, R. Ross, F. Kabbinavar, Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer, New England Journal of Medicine 350 (2004) 2335–2342.

[22] A. Sandler, R. Gray, M.C. Perry, J. Brahmer, J.H. Schiller, A. Dowlati, R. Lilenbaum, D.H. Johnson, Paclitaxel–carboplatin alone or with bevacizumab for non-small-cell lung cancer, New England Journal of Medicine 355 (2006) 2542–2550.

[23] K. Miller, M. Wang, J. Gralow, M. Dickler, M. Cobleigh, E.A. Perez, T. Shenkier, D. Cella, N.E. Davidson, Paclitaxel plus bevacizumab versus paclitaxel alone

[26] [27]

[28]

[29]

[30] [31]

[32] [33]

[34]

[35]

[36] [37] [38]

[39]

[40] [41]

[42]

[43]

[44]

[45]

135

for metastatic breast cancer, New England Journal of Medicine 357 (2007) 2666–2676.

J. Holash, S. Davis, N. Papadopoulos, S.D. Croll, L. Ho, M. Russell, P. Boland, R. Leidich, D. Hylton, E. Burova, E. Ioffe, T. Huang, C. Radziejewski, K. Bailey, J.P. Fandl, T. Daly, S.J. Wiegand, G.D. Yancopoulos, J.S. Rudge, VEGF-Trap: a VEGF blocker with potent antitumor effects, Proceedings of the National Academy of Sciences of the United States 99 (2002) 11393–11398.

L.M. Ellis, D.J. Hicklin, Pathways mediating resistance to vascular endothelial growth factor-targeted therapy, Clinical Cancer Research 14 (2008) 6371– 6375.

R.S. Kerbel, Tumor angiogenesis, New England Journal of Medicine 358 (2008) 2039–2049.

A.M. O’Farrell, T.J. Abrams, H.A. Yuen, T.J. Ngai, S.G. Louie, K.W. Yee, L.M. Wong, W. Hong, L.B. Lee, A. Town, B.D. Smolich, W.C. Manning, L.J. Murray, M.C. Heinrich, J.M. Cherrington, SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo, Blood 101 (2003) 3597– 3605.

S. Sleijfer, I. Ray-Coquard, Z. Papai, A. Le Cesne, M. Scurr, P. Schoffski, F. Collin, L. Pandite, S. Marreaud, A. De Brauwer, M. van Glabbeke, J. Verweij, J.Y. Blay, Pazopanib, a multikinase angiogenesis inhibitor, in patients with relapsed or refractory advanced soft tissue sarcoma: a phase II study from the European organisation for research and treatment of cancer-soft tissue and bone sarcoma group (EORTC study 62043), Journal of Clinical Oncology 27 (2009) 3126–3132.

P. Kupsch, B.F. Henning, K. Passarge, H. Richly, K. Wiesemann, R.A. Hilger, M.E. Scheulen, O. Christensen, E. Brendel, B. Schwartz, E. Hofstra, R. Voigtmann, S. Seeber, D. Strumberg, Results of a phase I trial of sorafenib (BAY 43-9006) in combination with oxaliplatin in patients with refractory solid tumors, including colorectal cancer, Clinical Colorectal Cancer 5 (2005) 188–196.

P.B. Langmuir, A. Yver, Vandetanib for the treatment of thyroid cancer, Clinical Pharmacology and Therapeutics 91 (2012) 71–80.

W.K. You, B. Sennino, C.W. Williamson, B. Falcon, H. Hashizume, L.C. Yao, D.T. Aftab, D.M. McDonald, VEGF and c-Met blockade amplify angiogenesis inhibition in pancreatic islet cancer, Cancer Research (2011).

T.H. Ho, E. Jonasch, Axitinib in the treatment of metastatic renal cell carcinoma, Future Oncology 7 (2011) 1247–1253.

F.A. Eskens, M.J. de Jonge, P. Bhargava, T. Isoe, M.M. Cotreau, B. Esteves, K. Hayashi, H. Burger, M. Thomeer, L. van Doorn, J. Verweij, Biologic and clinical activity of tivozanib (AV-951, KRN-951), a selective inhibitor of VEGF receptor-1, -2, and -3 tyrosine kinases, in a 4-week-on, 2-week-off schedule in patients with advanced solid tumors, Clinical Cancer Research 17 (2011) 7156–7163.

F. Jiang, D.H. Albert, Y. Luo, P. Tapang, K. Zhang, S.K. Davidsen, G.B. Fox, R. Lesniewski, E.M. McKeegan, ABT-869, a multitargeted receptor tyrosine kinase inhibitor, reduces tumor microvascularity and improves vascular wall integrity in preclinical tumor models, Journal of Pharmacology and Experimental Therapeutics 338 (2011) 134–142.

E.H. Tan, G.D. Goss, R. Salgia, B. Besse, D.R. Gandara, N.H. Hanna, J.C. Yang, R. Thertulien, M. Wertheim, J. Mazieres, T. Hensing, C. Lee, N. Gupta, R. Pradhan, J. Qian, Q. Qin, F.A. Scappaticci, J.L. Ricker, D.M. Carlson, R.A. Soo, Phase 2 trial of Linifanib (ABT-869) in patients with advanced non-small cell lung cancer, Journal of Thoracic Oncology 6 (2011) 1418–1425.

R.K. Jain, Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy, Science 307 (2005) 58–62.

UNIO Health, US National Institute of Health. <http://clinicaltrials.gov/>.

M.M. Cooney, W. van Heeckeren, S. Bhakta, J. Ortiz, S.C. Remick, Drug insight: vascular disrupting agents and angiogenesis-novel approaches for drug delivery, Nature Clinical Practice Oncology 3 (2006) 682–692.

M.J. McKeage, J. Von Pawel, M. Reck, M.B. Jameson, M.A. Rosenthal, R. Sullivan, D. Gibbs, P.N. Mainwaring, M. Serke, J.J. Lafitte, C. Chouaid, L. Freitag, E. Quoix, Randomised phase II study of ASA404 combined with carboplatin and paclitaxel in previously untreated advanced non-small cell lung cancer, British Journal of Cancer 99 (2008) 2006–2012.

G. Bergers, D. Hanahan, Modes of resistance to anti-angiogenic therapy, Nature Reviews Cancer 8 (2008) 592–603.

F. Shojaei, X. Wu, A.K. Malik, C. Zhong, M.E. Baldwin, S. Schanz, G. Fuh, H.P. Gerber, N. Ferrara, Tumor refractoriness to anti-VEGF treatment is mediated

by CD11b + Gr1+ myeloid cells, Nature Biotechnology 25 (2007) 911–920.

F. Shojaei, J.H. Lee, B.H. Simmons, A. Wong, C.O. Esparza, P.A. Plumlee, J. Feng, A.E. Stewart, D.D. Hu-Lowe, J.G. Christensen, HGF/c-Met acts as an alternative angiogenic pathway in sunitinib-resistant tumors, Cancer Research 70 (2010) 10090–10100.

H.P. Gerber, X. Wu, L. Yu, C. Wiesmann, X.H. Liang, C.V. Lee, G. Fuh, C. Olsson, L. Damico, D. Xie, Y.G. Meng, J. Gutierrez, R. Corpuz, B. Li, L. Hall, L. Rangell, R. Ferrando, H. Lowman, F. Peale, N. Ferrara, Mice expressing a humanized form of VEGF-A may provide insights into the safety and efficacy of anti-VEGF antibodies, Proceedings of the National Academy of Sciences of the United States 104 (2007) 3478–3483.

H.J. Broxterman, J. Lankelma, K. Hoekman, Resistance to cytotoxic and antiangiogenic anticancer agents: similarities and differences, Drug Resistance Updates 6 (2003) 111–127.

Q. Pan, Y. Chanthery, W.C. Liang, S. Stawicki, J. Mak, N. Rathore, R.K. Tong, J. Kowalski, S.F. Yee, G. Pacheco, S. Ross, Z. Cheng, J. Le Couter, G. Plowman, F. Peale, A.W. Koch, Y. Wu, A. Bagri, M. Tessier-Lavigne, R.J. Watts, Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth, Cancer Cell 11 (2007) 53–67.

136

F. Shojaei / Cancer Letters 320 (2012) 130–137

[46] P. Mancuso, M. Colleoni, A. Calleri, L. Orlando, P. Maisonneuve, G. Pruneri, A. Agliano, A. Goldhirsch, Y. Shaked, R.S. Kerbel, F. Bertolini, Circulating endothelial-cell kinetics and viability predict survival in breast cancer patients receiving metronomic chemotherapy, Blood 108 (2006) 452–459.

[47] O. Casanovas, D.J. Hicklin, G. Bergers, D. Hanahan, Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors, Cancer Cell 8 (2005) 299–309.

[48] M. Chiao, Y. Yang, W. Cheng, C. Shen, J. Ko, CD133+ glioblastoma stem-like cells induce vascular mimicry in vivo, Current Neurovascular Research 1 (2011) 210–219.

[49] L. Ricci-Vitiani, R. Pallini, M. Biffoni, M. Todaro, G. Invernici, T. Cenci, G. Maira, E.A. Parati, G. Stassi, L.M. Larocca, R. De Maria, Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells, Nature 468 (2010) 824–828.

[50] R. Wang, K. Chadalavada, J. Wilshire, U. Kowalik, K.E. Hovinga, A. Geber, B. Fligelman, M. Leversha, C. Brennan, V. Tabar, Glioblastoma stem-like cells give rise to tumour endothelium, Nature 468 (2010) 829–833.

[51] C. Mailhos, U. Modlich, J. Lewis, A. Harris, R. Bicknell, D. Ish-Horowicz, Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis, Differentiation 69 (2001) 135–144.

[52] I. Noguera-Troise, C. Daly, N.J. Papadopoulos, S. Coetzee, P. Boland, N.W. Gale, H.C. Lin, G.D. Yancopoulos, G. Thurston, Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis, Nature 444 (2006) 1032– 1037.

[53] J. Ridgway, G. Zhang, Y. Wu, S. Stawicki, W.C. Liang, Y. Chanthery, J. Kowalski, R.J. Watts, C. Callahan, I. Kasman, M. Singh, M. Chien, C. Tan, J.A. Hongo, F. de Sauvage, G. Plowman, M. Yan, Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis, Nature 444 (2006) 1083–1087.

[54] T. Cascone, M.H. Herynk, L. Xu, Z. Du, H. Kadara, M.B. Nilsson, C.J. Oborn, Y.Y. Park, B. Erez, J.J. Jacoby, J.S. Lee, H.Y. Lin, F. Ciardiello, R.S. Herbst, R.R. Langley, J.V. Heymach, Upregulated stromal EGFR and vascular remodeling in mouse xenograft models of angiogenesis inhibitor-resistant human lung adenocarcinoma, Journal of Clinical Investigation 121 (2011) 1313–1328.

[55] F. Shojaei, C. Zhong, X. Wu, L. Yu, N. Ferrara, Role of myeloid cells in tumor angiogenesis and growth, Trends in Cell Biology 18 (2008) 372–378.

[56] F. Shojaei, X. Wu, X. Qu, M. Kowanetz, L. Yu, M. Tan, Y.G. Meng, N. Ferrara, GCSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models, Proceedings of the National Academy of Sciences of the United States 106 (2009) 6742–6747.

[57] M. Kowanetz, X. Wu, J. Lee, M. Tan, T. Hagenbeek, X. Qu, L. Yu, J. Ross, N. Korsisaari, T. Cao, H. Bou-Reslan, D. Kallop, R. Weimer, M.J. Ludlam, J.S. Kaminker, Z. Modrusan, N. van Bruggen, F.V. Peale, R. Carano, Y.G. Meng, N. Ferrara, Granulocyte-colony stimulating factor promotes lung metastasis through mobilization of Ly6G + Ly6C + granulocytes, Proceedings of the National Academy of Sciences of the United States 107 (2010) 21248–21255.

[58] H. Nozawa, C. Chiu, D. Hanahan, Infiltrating neutrophils mediate the initial angiogenic switch in a mouse model of multistage carcinogenesis, Proceedings of the National Academy of Sciences of the United States 103 (2006) 12493–12498.

[59] B. Huang, P.Y. Pan, Q. Li, A.I. Sato, D.E. Levy, J. Bromberg, C.M. Divino, S.H. Chen, Gr-1 + CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host, Cancer Research 66 (2006) 1123–1131.

[60] Y. Crawford, I. Kasman, L. Yu, C. Zhong, X. Wu, Z. Modrusan, J. Kaminker, N. Ferrara, PDGF-C mediates the angiogenic and tumorigenic properties of fibroblasts associated with tumors refractory to anti-VEGF treatment, Cancer Cell 15 (2009) 21–34.

[61] D. von Tell, A. Armulik, C. Betsholtz, Pericytes and vascular stability, Experimental Cell Research 312 (2006) 623–629.

[62] M. Paez-Ribes, E. Allen, J. Hudock, T. Takeda, H. Okuyama, F. Vinals, M. Inoue, G. Bergers, D. Hanahan, O. Casanovas, Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis, Cancer Cell 15 (2009) 220–231.

[63] A.K. Lucio-Eterovic, Y. Piao, J.F. de Groot, Mediators of glioblastoma resistance and invasion during antivascular endothelial growth factor therapy, Clinical Cancer Research 15 (2009) 4589–4599.

[64] J.M. Ebos, C.R. Lee, W. Cruz-Munoz, G.A. Bjarnason, J.G. Christensen, R.S. Kerbel, Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis, Cancer Cell 15 (2009) 232–239.

[65] F. Shojaei, B.H. Simmons, J.H. Lee, B.L. Patrick, J.G. Christensen, HGF/c-met pathway is one of the mediators of sunitinib-induced tumor cell typedependent metastasis, Cancer Letters (2012) [Epub ahead of print].

[66] A. Rapisarda, G. Melillo, Role of the hypoxic tumor microenvironment in the resistance to anti-angiogenic therapies, Drug Resistance Updates 12 (2009) 74–80.

[67] J.M. Ebos, C.R. Lee, J.G. Christensen, A.J. Mutsaers, R.S. Kerbel, Multiple circulating proangiogenic factors induced by sunitinib malate are tumorindependent and correlate with antitumor efficacy, Proceedings of the National Academy of Sciences of the United States 104 (2007) 17069–17074.

[68] X. Xian, J. Hakansson, A. Stahlberg, P. Lindblom, C. Betsholtz, H. Gerhardt, H. Semb, Pericytes limit tumor cell metastasis, Journal of Clinical Investigation 116 (2006) 642–651.

[69] J.M. Ebos, R.S. Kerbel, Antiangiogenic therapy: impact on invasion, disease progression, and metastasis, Nature Reviews Clinical Oncology 8 (2011) 210– 221.

[70] A. Gerger, M. LaBonte, H.J. Lenz, Molecular predictors of response to antiangiogenesis therapies, Cancer Journal 17 (2011) 134–141.

[71] S.X. Yang, S.M. Steinberg, D. Nguyen, T.D. Wu, Z. Modrusan, S.M. Swain, Gene expression profile and angiogenic marker correlates with response to neoadjuvant bevacizumab followed by bevacizumab plus chemotherapy in breast cancer, Clinical Cancer Research 14 (2008) 5893–5899.

[72] T.K. Choueiri, M.M. Regan, J.E. Rosenberg, W.K. Oh, J. Clement, A.M. Amato, D. McDermott, D.C. Cho, M.B. Atkins, S. Signoretti, Carbonic anhydrase IX and pathological features as predictors of outcome in patients with metastatic clear-cell renal cell carcinoma receiving vascular endothelial growth factortargeted therapy, BJU International 106 (2010) 772–778.

[73] T.T. Batchelor, A.G. Sorensen, E. di Tomaso, W.T. Zhang, D.G. Duda, K.S. Cohen, K.R. Kozak, D.P. Cahill, P.J. Chen, M. Zhu, M. Ancukiewicz, M.M. Mrugala, S. Plotkin, J. Drappatz, D.N. Louis, P. Ivy, D.T. Scadden, T. Benner, J.S. Loeffler, P.Y. Wen, R.K. Jain, AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients, Cancer Cell 11 (2007) 83–95.

[74] Y. Lin, D.J. Weisdorf, A. Solovey, R.P. Hebbel, Origins of circulating endothelial cells and endothelial outgrowth from blood, Journal of Clinical Investigation 105 (2000) 71–77.

[75] J.L. Rowand, G. Martin, G.V. Doyle, M.C. Miller, M.S. Pierce, M.C. Connelly, C. Rao, L.W. Terstappen, Endothelial cells in peripheral blood of healthy subjects and patients with metastatic carcinomas, Cytometry A 71 (2007) 105–113.

[76] S. Dellapasqua, F. Bertolini, V. Bagnardi, E. Campagnoli, E. Scarano, R. Torrisi, Y. Shaked, P. Mancuso, A. Goldhirsch, A. Rocca, E. Pietri, M. Colleoni, Metronomic cyclophosphamide and capecitabine combined with bevacizumab in advanced breast cancer, Journal of Clinical Oncology 26 (2008) 4899–4905.

[77] R.K. Jain, D.G. Duda, C.G. Willett, D.V. Sahani, A.X. Zhu, J.S. Loeffler, T.T. Batchelor, A.G. Sorensen, Biomarkers of response and resistance to antiangiogenic therapy, Nature Reviews Clinical Oncology 6 (2009) 327–338.

[78] T.G. Gorgels, J.H. Waarsing, M. Herfs, D. Versteeg, F. Schoensiegel, T. Sato, R.O. Schlingemann, B. Ivandic, C. Vermeer, L.J. Schurgers, A.A. Bergen, Vitamin K supplementation increases vitamin K tissue levels but fails to counteract ectopic calcification in a mouse model for pseudoxanthoma elasticum, Journal of Molecular Medicine (Berlin) 89 (2011) 1125–1135.

[79] F. Loupakis, A. Falcone, G. Masi, A. Fioravanti, R.S. Kerbel, M. Del Tacca, G. Bocci, Vascular endothelial growth factor levels in immunodepleted plasma of cancer patients as a possible pharmacodynamic marker for bevacizumab activity, Journal of Clinical Oncology 25 (2007) 1816–1818.

[80] M.S. Gordon, K. Margolin, M. Talpaz, G.W. Sledge Jr., E. Holmgren, R. Benjamin, S. Stalter, S. Shak, D. Adelman, Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer, Journal of Clinical Oncology 19 (2001) 843–850.

[81] G.C. Jayson, C. Mulatero, M. Ranson, J. Zweit, A. Jackson, L. Broughton, J. Wagstaff, L. Hakansson, G. Groenewegen, J. Lawrance, M. Tang, L. Wauk, D. Levitt, S. Marreaud, F.F. Lehmann, M. Herold, H. Zwierzina, Phase I investigation of recombinant anti-human vascular endothelial growth factor antibody in patients with advanced cancer, European Journal of Cancer 41 (2005) 555–563.

[82] N. Denduluri, S.X. Yang, A.W. Berman, D. Nguyen, D.J. Liewehr, S.M. Steinberg, S.M. Swain, Circulating biomarkers of bevacizumab activity in patients with breast cancer, Cancer Biology & Therapy 7 (2008) 15–20.

[83] C. Porta, C. Paglino, M. De Amici, S. Quaglini, L. Sacchi, I. Imarisio, C. Canipari, Predictive value of baseline serum vascular endothelial growth factor and neutrophil gelatinase-associated lipocalin in advanced kidney cancer patients receiving sunitinib, Kidney International 77 (2010) 809–815.

[84] L. Xu, D.G. Duda, E. di Tomaso, M. Ancukiewicz, D.C. Chung, G.Y. Lauwers, R. Samuel, P. Shellito, B.G. Czito, P.C. Lin, M. Poleski, R. Bentley, J.W. Clark, C.G. Willett, R.K. Jain, Direct evidence that bevacizumab, an anti-VEGF antibody, up-regulates SDF1alpha, CXCR4, CXCL6, and neuropilin 1 in tumors from patients with rectal cancer, Cancer Research 69 (2009) 7905–7910.

[85] M.L. Maitland, C. Grimsley, H. Kuttab-Boulos, D. Witonsky, K.E. Kasza, L. Yang, B.A. Roe, A. Di Rienzo, Comparative genomics analysis of human sequence variation in the UGT1A gene cluster, Pharmacogenomics Journal 6 (2006) 52– 62.

[86] M. Scartozzi, E. Galizia, S. Chiorrini, R. Giampieri, R. Berardi, C. Pierantoni, S. Cascinu, Arterial hypertension correlates with clinical outcome in colorectal cancer patients treated with first-line bevacizumab, Annals of Oncology 20 (2009) 227–230.

[87] B.P. Schneider, M. Wang, M. Radovich, G.W. Sledge, S. Badve, A. Thor, D.A. Flockhart, B. Hancock, N. Davidson, J. Gralow, M. Dickler, E.A. Perez, M. Cobleigh, T. Shenkier, S. Edgerton, K.D. Miller, Association of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 genetic polymorphisms with outcome in a trial of paclitaxel compared with paclitaxel plus bevacizumab in advanced breast cancer: ECOG 2100, Journal of Clinical Oncology 26 (2008) 4672–4678.

[88] O.M. Hahn, C. Yang, M. Medved, G. Karczmar, E. Kistner, T. Karrison, E. Manchen, M. Mitchell, M.J. Ratain, W.M. Stadler, Dynamic contrast-enhanced magnetic resonance imaging pharmacodynamic biomarker study of sorafenib in metastatic renal carcinoma, Journal of Clinical Oncology 26 (2008) 4572– 4578.

[89] B. Morgan, A.L. Thomas, J. Drevs, J. Hennig, M. Buchert, A. Jivan, M.A. Horsfield, K. Mross, H.A. Ball, L. Lee, W. Mietlowski, S. Fuxuis, C. Unger, K. O’Byrne, A. Henry, G.R. Cherryman, D. Laurent, M. Dugan, D. Marme, W.P.

F. Shojaei / Cancer Letters 320 (2012) 130–137

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

Steward, Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: results from two phase I studies, Journal of Clinical Oncology 21 (2003) 3955– 3964.

W.B. Nagengast, M.N. Lub-de Hooge, S.F. Oosting, W.F. den Dunnen, F.J. Warnders, A.H. Brouwers, J.R. de Jong, P.M. Price, H. Hollema, G.A. Hospers, P.H. Elsinga, J.W. Hesselink, J.A. Gietema, E.G. de Vries, VEGF-PET imaging is a noninvasive biomarker showing differential changes in the tumor during sunitinib treatment, Cancer Research 71 (2011) 143–153.

T.F. Hansen, R.D. Christensen, R.F. Andersen, K.L. Spindler, A. Johnsson, A. Jakobsen, The predictive value of single nucleotide polymorphisms in the VEGF system to the efficacy of first-line treatment with bevacizumab plus chemotherapy in patients with metastatic colorectal cancer: results from the Nordic ACT trial, The International Journal of Colorectal Disease (2011).

J. Garcia-Donas, E. Esteban, L.J. Leandro-Garcia, D.E. Castellano, A.G. del Alba, M.A. Climent, J.A. Arranz, E. Gallardo, J. Puente, J. Bellmunt, B. Mellado, E. Martinez, F. Moreno, A. Font, M. Robledo, C. Rodriguez-Antona, Single nucleotide polymorphism associations with response and toxic effects in patients with advanced renal-cell carcinoma treated with first-line sunitinib: a multicentre, observational, prospective study, Lancet Oncology 12 (2011) 1143–1150.

F. Shojaei, M. Singh, J.D. Thompson, N. Ferrara, Role of Bv8 in neutrophildependent angiogenesis in a transgenic model of cancer progression, Proceedings of the National Academy of Sciences of the United States 105 (2008) 2640–2645.

F. Shojaei, X. Wu, C. Zhong, L. Yu, X.H. Liang, J. Yao, D. Blanchard, C. Bais, F.V. Peale, N. van Bruggen, C. Ho, J. Ross, M. Tan, R.A. Carano, Y.G. Meng, N. Ferrara, Bv8 regulates myeloid-cell-dependent tumour angiogenesis, Nature 450 (2007) 825–831.

R. Hadj-Slimane, Y. Lepelletier, N. Lopez, C. Garbay, F. Raynaud, Short interfering RNA (siRNA), a novel therapeutic tool acting on angiogenesis, Biochimie 89 (2007) 1234–1244.

S. Anand, B.K. Majeti, L.M. Acevedo, E.A. Murphy, R. Mukthavaram, L. Scheppke, M. Huang, D.J. Shields, J.N. Lindquist, P.E. Lapinski, P.D. King, S.M. Weis, D.A. Cheresh, MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis, Nature Medicine 16 (2010) 909–914.

M.R. Barakat, P.K. Kaiser, VEGF inhibitors for the treatment of neovascular age-related macular degeneration, Expert Opinion on Investigational Drugs 18 (2009) 637–646.

N. Ferrara, Vascular endothelial growth factor and age-related macular degeneration: from basic science to therapy, Nature Medicine 16 (2010) 1107–1111.

A.A. Epenetos, D. Snook, H. Durbin, P.M. Johnson, J. Taylor-Papadimitriou, Limitations of radiolabeled monoclonal antibodies for localization of human neoplasms, Cancer Research 46 (1986) 3183–3191.

K. Yoncheva, G. Momekov, Antiangiogenic anticancer strategy based on nanoparticulate systems, Expert Opinion on Drug Delivery 8 (2011) 1041– 1056.

L. Li, C.A. Wartchow, S.N. Danthi, Z. Shen, N. Dechene, J. Pease, H.S. Choi, T. Doede, P. Chu, S. Ning, D.Y. Lee, M.D. Bednarski, S.J. Knox, A novel antiangiogenesis therapy using an integrin antagonist or anti-Flk-1 antibody coated 90Y-labeled nanoparticles, International Journal of Radiation Oncology, Biology, Physics 58 (2004) 1215–1227.

A.S. Abu Lila, T. Ishida, H. Kiwada, Targeting anticancer drugs to tumor vasculature using cationic liposomes, Pharmaceutical Research 27 (2010) 1171–1183.

137

[103] A.S. Abu Lila, S. Kizuki, Y. Doi, T. Suzuki, T. Ishida, H. Kiwada, Oxaliplatin encapsulated in PEG-coated cationic liposomes induces significant tumor growth suppression via a dual-targeting approach in a murine solid tumor model, Journal of Controlled Release 137 (2009) 8–14.

[104] K. Temming, R.M. Schiffelers, G. Molema, R.J. Kok, RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature, Drug Resistance Updates 8 (2005) 381–402.

[105] E.A. Murphy, B.K. Majeti, L.A. Barnes, M. Makale, S.M. Weis, K. Lutu-Fuga, W. Wrasidlo, D.A. Cheresh, Nanoparticle-mediated drug delivery to tumor vasculature suppresses metastasis, Proceedings of the National academy of Sciences of the United States of America 105 (2008) 9343–9348.

[106] M.S. Iordanov, B.E. Magun, Different mechanisms of c-Jun NH(2)-terminal kinase-1 (JNK1) activation by ultraviolet-B radiation and by oxidative stressors, Journal of Biological Chemistry 274 (1999) 25801–25806.

[107] M. Backer, J. Backer, Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of Shiga-like toxin-VEGF fusion proteins, Bioconjugate Chemistry 12 (2001) 1066–1073.

[108] M.V. Backer, C.V. Hamby, J.M. Backer, Inhibition of vascular endothelial growth factor receptor signaling in angiogenic tumor vasculature, Advances in Genetics 67 (2009) 1–27.

[109] B.A. Teicher, Antibody-drug conjugate targets, Current Cancer Drug Targets 9 (2009) 982–1004.

[110] H.P. Gerber, P.D. Senter, I.S. Grewal, Antibody drug-conjugates targeting the tumor vasculature: current and future developments, MAbs 1 (2009) 247– 253.

[111] L. Borsi, E. Balza, M. Bestagno, P. Castellani, B. Carnemolla, A. Biro, A. Leprini, J. Sepulveda, O. Burrone, D. Neri, L. Zardi, Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin, International Journal of Cancer 102 (2002) 75–85.

[112] K.S. Midwood, T. Hussenet, B. Langlois, G. Orend, Advances in tenascin-C biology, Cellular and Molecular Life Sciences 68 (2011) 3175–3199.

[113] A.A. van der Veldt, M.R. Meijerink, A.J. van den Eertwegh, A. Bex, G. de Gast, J.B. Haanen, E. Boven, Sunitinib for treatment of advanced renal cell cancer: primary tumor response, Clinical Cancer Research 14 (2008) 2431–2436.

[114] J.K. Smith, N.M. Mamoon, R.J. Duhe, Emerging roles of targeted small molecule protein-tyrosine kinase inhibitors in cancer therapy, Oncology Research 14 (2004) 175–225.

[115] M.A. Fabian, W.H. Biggs 3rd, D.K. Treiber, C.E. Atteridge, M.D. Azimioara, M.G. Benedetti, T.A. Carter, P. Ciceri, P.T. Edeen, M. Floyd, J.M. Ford, M. Galvin, J.L. Gerlach, R.M. Grotzfeld, S. Herrgard, D.E. Insko, M.A. Insko, A.G. Lai, J.M. Lelias, S.A. Mehta, Z.V. Milanov, A.M. Velasco, L.M. Wodicka, H.K. Patel, P.P. Zarrinkar, D.J. Lockhart, A small molecule-kinase interaction map for clinical kinase inhibitors, Nature Biotechnology 23 (2005) 329–336.

[116] R.C. Kane, A.T. Farrell, H. Saber, S. Tang, G. Williams, J.M. Jee, C. Liang, B. Booth, N. Chidambaram, D. Morse, R. Sridhara, P. Garvey, R. Justice, R. Pazdur, Sorafenib for the treatment of advanced renal cell carcinoma, Clinical Cancer Research 12 (2006) 7271–7278.

[117] L. Lang, FDA approves sorafenib for patients with inoperable liver cancer, Gastroenterology 134 (2008) 379.

[118] S.A. Wells Jr., J.E. Gosnell, R.F. Gagel, J. Moley, D. Pfister, J.A. Sosa, M. Skinner, A. Krebs, J. Vasselli, M. Schlumberger, Vandetanib for the treatment of patients with locally advanced or metastatic hereditary medullary thyroid cancer, Journal of Clinical Oncology 28 (2010) 767–772.

[119] F.M. Yakes, J. Chen, J. Tan, K. Yamaguchi, Y. Shi, P. Yu, F. Qian, F. Chu, F. Bentzien, B. Cancilla, J. Orf, A. You, A.D. Laird, S. Engst, L. Lee, J. Lesch, Y.C. Chou, A.H. Joly, Cabozantinib (XL184), a novel MET and VEGFR2 inhibitor, simultaneously suppresses metastasis, angiogenesis, and tumor growth, Molecular Cancer Therapeutics 10 (2011) 2298–2308.