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1 Prostate Tumor Microenvironment, Hypoxia and Tumor Neoangiogenesis

  • Stefania Staibano 

  1. Department of Advanced Biomedical Sciences, Pathology Section Faculty of Medicine and Surgery, University of Naples “Federico II”, via S. Pansini, n.5, Naples, Italy

    Stefania Staibano

Prostate cancer-associated angiogenesis is a well recognised process (Russo et al. 2012).

Microvessel density (MVD) usually is higher in primary tumors of patients with metastatic disease compared with localised prostate cancers (Weidner et al. 1993; Fregene et al. 1993; Strohmeyer et al. 2000; Gravdal et al. 2009). As well, higher MVD correlates with advanced pathological stage (Lee et al. 2004), increased PSA levels (Lee et al. 2001a, b), higher tumor grade (Park et al. 2007), increased metastatic potential (Aragon-Ching and Dahut 2009; Park et al. 2007), and decreased survival of patients (Park et al. 2007; Lee et al. 2001a, b).

Moreover, tumor blood vessels show multiple structural and functional abnormalities (Russo et al. 2012), increased tortuosity, blind ends and high cellular proliferation rate, leading to dysfunctional and heterogeneous tumor tissue microcirculation, with frequent avascular tumor areas, hypoxia, acidosis, and glucose deprivation (Shannon et al. 2003; Teicher et al. 1990; Vaupel et al. 1989; Airley et al. 2000; Brown 1999; Folkman 1971).

This results in a net efflux of fluid into the interstitial space, devoted of functional lymphatics, so that it distends the extracellular matrix and increases the interstitial pressure (An et al. 1998).

All these features are associated with metastatic risk (Siim et al. 1996; Wang et al. 1992; Peters et al. 2001).

VEGF is a 46-kDa dimeric protein also known as a vascular permeability factor (VPF), and represents the most potent growth factor acting in stimulating cell proliferation, angiogenesis and lymphangiogenesis (Ferrer et al. 1998; Shweiki et al. 1995). VEGF, in prostate cancer progression, is regulated by hypoxia (Shweiki et al. 1992, 1995; Minchenko et al. 1994; Walker et al. 1994), cytokines, and androgens; moreover, several oncogenes, as Ras-, Raf-, and Src, the inactivation of tumor-suppressor genes as p53 and von Hippel – Lindau (Ravi et al. 2000) concur to its modulation.

VEGF immunohistochemical expression is highest in metastatic prostatic cancer tissue, but it does not predict prostate cancer progression (Botelho et al. 2010), nor correlates with VEGF expression and clinical and pathological features of tumors (Shariat et al. 2004).

VEGF acts in a paracrine manner by binding its receptors (VEGF-R1 and VEGF-R2) expressed on the surface of endothelial cells. VEGF binding activates the receptor’s tyrosine kinase activity, and via the stimulation of several molecular pathways, as ERK and Akt, leads to vasodilatation, increased vascular permeability, cell proliferation, degradation and invasion of the underlying stroma (Aragon-Ching and Dahut 2009).

However, these vascular growth factors have multiple functions. As an example, prostate cancer cells overexpress also the VEGF receptor (VEGFR), so VEGF and VEGFR reciprocally act in an autocrine manner promoting, besides neo-angiogenesis, prostate cancer cell proliferation and survival (Jackson et al. 2002). In addition, it inhibits tumor cell apoptosis by inducing the expression of the anti-apoptotic protein Bcl-2 (Pidgeon et al. 2001).

Addition of VEGF inhibitors to antiandrogen therapy results in increased oxygen delivery to hypoxic tumors areas and thus further potentiates radiation therapy (Zhu and Kyprianou 2008).

Prostate tumor cells respond to hypoxia with the over-transcription of the hypoxia-inducible factor-1a (HIF-1) (Rang et al. 1999), which in turn overstimulates VEGF production and leads to neo-angiogenesis (Cvetkovic et al. 2001). VEGF production and signaling is partly dependent on mTOR induced expression of HIF-1a (Treins et al. 2002).

HIF-1a is hydroxylated at the proline residue and degraded by interaction with the von Hipple-Lindau protein complex and proteosome machinery (Semenza 2003; Forsythe et al. 1996) in normoxic conditions. In prostate cells, androgens can activate HIF-1 through an autocrine loop, and HIF-1 interacts with AR on PSA gene promoter, thereby activating its expression (Zhong et al. 2008).

Under hypoxic conditions, as in advanced prostate cancer, HIF-1a protein is stabilized and translocated into the nucleus for specific gene expression regulation including VEGF, and regulates intracellular pH, metabolism, cell invasion and autophagy, preventing death of aggressive cancer cells (Pouyssegur et al. 2006).

HIF-1a is then a preferential target for the development of anticancer drugs (Pili and Donehower 2003).

Histone deacetylase (HDAC) inhibitors have shown an anti-angiogenic activity mediated in part by HIF-1a down-regulation in both tumor and endothelial cells, with the consequent down-regulation of VEGF and other HIF-1a regulated angiogenesis-related genes (Qian et al. 2004). Class II HDAC are important modifiers of HIF-1a. Recently, it has been reported that the HDAC inhibitor LBH589 reduced tumor growth and angiogenesis in a preclinical prostate cancer model (Qian et al. 2006a, b).

Prostate cancer cells overexpress also TGF β, which promotes either extracellular matrix production and angiogenesis (Russell et al. 1998), favouring also osteoblastic bone metastases in experimental systems. The increase of TGF β RI is associated with high-grade and higher clinical stage of prostate cancer.

TGFβ RI expression correlates with tumor vascularity, tumor grade, and metastasis (Wikström et al. 2001). On the opposite side, TGF β RIII expression is decreased or lost in most human prostate cancers, where it correlates with advanced tumor stage and high risk of PSA recurrence.

The occurrence of intraepithelial prostate cancer correlates instead with the loss of TGF β RII responsiveness in stromal fibroblasts. Thus, partially blocking TGFβ through angiogenesis inhibitors, e.g. angiostatin and endostatin could potentially reverse the continuous stimulation of tumor angiogenesis (Deryugina and Quigley 2006).

Prostate cancer has the ability to produce MMPs, TGFβ; and cyclooxygenase 2 (COX-2).

Several endogenous inhibitors of angiogenesis have also been described in prostate cancer, namely angiostatin, endostatin, PSA, TSP1, interleukin 8, and interferons.

Overall, the microenvironment of prostate cancer is a critical determinant in cancer genesis (Chung et al. 2005).

2 Targeting the Angiogenic Pathways in Castration-resistant Prostate Cancer

  • Paolo Antonio Ascierto 

  1. Unit of Melanoma, Cancer Immunotherapy and Innovative Therapy, Department of Melanoma, National Institute of Tumors Fondazione “G. Pascale”, Via Mariano Semmola 1, 80131, Naples, Italy

    Paolo Antonio Ascierto

Monoclonal antibody Bevacizumab is a recombinant humanized IgG1 monoclonal antibody with high affinity and specificity for all VEGF-A isoforms. Upon binding to soluble VEGF-A, bevacizumab limits ligand binding to EC receptors VEGFR-1 and VEGFR-2, thus blocking pro-angiogenic intracellular signals transduction.

In a phase II study, Reese et al. evaluated bevacizumab at 10 mg/kg every 14 days for 6 cycles in 15 chemotherapy-naïve patients with CRPC. No objective responses were showed.

But there was a PSA decline (less than 50 %) in 27 % of patients. Antibodies to VEGF slow tumor proliferation in prostate cancer xenograft models, especially when combined with chemotherapy (Gross et al. 2009; Antonarakis and Armstrong 2011).

Despite strong preclinical rationale, a phase III randomized study in men with chemotherapy-untreated CRPC (CALGB 90401) failed to show a survival advantage with the anti-VEGF antibody bevacizumab when combined with docetaxel compared with docetaxel used alone (22.6 vs 21.5 months), although significant improvements were seen with respect to PSA responses (70 vs 58 %) and radiographic responses (53 vs 42 %), as well as progression-free survival (9.9 vs 7.5 months) (George et al. 2011).

However, these results do not indicate that antiangiogenic therapies may never have a role in the treatment of CRPC, as much of this failure may be explained by an imbalance of treatment-related toxicities (cardiovascular events, neutropenic complications) in this older population with multiple co-morbidities. To this end, it was reported that the presence and number of co-morbidities (for example, cardiovascular disease, hypertension, diabetes, renal disease, liver disease) among patients in the CALGB 90401 trial significantly correlated with survival, and that there was an increase in the average number of co-morbidities in the docetaxel-bevacizumab arm (Wu et al. 2005).

Future development of this and other antiangiogenic agents may rely on combinations with other classes of angiogenesis inhibitors or other chemotherapeutic drugs whose toxicities do not overlap, and will require careful patient selection for those men most likely to benefit and not be harmed by this class of agents.

Combinations of bevacizumab with other agents were also evaluated. The phase II trial CALGB 90006 enrolled 79 patients with metastatic CRPC patients who were treated with docetaxel (70 mg/mq every 21 days), bevacizumab (15 mg/kg every 21 days) and estramustine (280 mg on days 1–5 of the 21 day cycle).

Promising results were showed with a median PFS of 8 months and a median OS of 24 months, with a PSA decline (higher than 50 %) in 75 % of patients (epub.theoncologist.com) (Sturge et al. 2011).

In a phase III trial with metastatic CRPC, 1,050 patients were randomized to receive docetaxel (75 mg/mq every 21 days), prednisone (5 mg twice daily) and either bevacizumab (15 mg/kg every 21 days) or placebo.

This study showed, despite an improvement in the secondary endpoints of progression-free survival (PFS), measurable disease response and post-therapy PSA decline, but the combination with bevacizumab was not statistically significant for OS (22.6 vs 21.5 months). Furthermore, there was higher toxicity in the experimental arm (epub.theoncologist.com).

Other combinations of bevacizumab with other drugs (cytotoxin agents and immunotherapy) didn’t show results and further studies are needed. Sunitinib is an oral TKI targeted to all three VEGFR isoforms as well as PDGFβ and KIT, currently approved for renal cell carcinoma (Powles et al. 2011) and gastrointestinal stromal tumor. Several phase II studies of sunitinib were conducted in patients with metastatic CRPC, both in chemotherapy-naïve patients and in post-docetaxel setting (epub.theoncologist.com).

Also a multicenter, randomized, double-blind phase III trial comparing sunitinib plus prednisone versus prednisone alone (NCT00676650) in patients with post docetaxel progressive metastatic CRPC was conducted. But This trial has been recently interrupted prematurely since the combination of sunitinib with prednisone didn’t improve OS when compared to prednisone alone.

Aflibercept is a recombinant fusion protein of the extracellular domain of human VEGF-R1 and VEGF-R2 and the Fc portion of human IgG. It acts as a ‘VEGF trap’ or decoy receptor, binding free ligand and preventing it from interacting with and activating membrane-bound receptor. As expected, it potently binds all naturally occurring ligands of VEGF-R1 and VEGF-R2, including VEGF-A, VEGF-B, and placental growth factor, and so may be anticipated to possess greater anti-angiogenic activity than bevacizumab. In phase I trials, the combination of aflibercept and docetaxel was shown to be safe and well tolerated, and the combination is now under evaluation in a large phase III trial (VENICE study) in patients with mCRPC.51 This study has completed accrual of approximately 1,200 patients, and is expected to report in mid-2012, with overall survival as the primary endpoint (Corcoran and Gleave 2012).

Other immunomodulatory-antiangiogenic agents like Thalidomide have been studied. The best data on thalidomide were in combination with other cytotoxic agents. Weekly docetaxel (30 mg/mq weekly for 3 out every 4 weeks) with or without thalidomide (200 mg/day) has been evaluated in chemotherapy-naïve metastatic CRPC. The combination arm was favored in terms of PSA decline (53 versus 37 % experiencing >50 % decrease in PSA) and PFS (5.9 versus 3.7 months). The most frequent adverse events were fatigue, peripheral neuropathy and constipation. Furthermore the combination arm may increase thromboembolic events and requires prophylactic anticoagulant therapy. Lenalidomide is an analog of thalidomide that has been evaluated in CRPC patients showing lower toxicity than thalidomide and a better antiangiogenic effect (Merino et al. 2011).

A phase I–II trial evaluating efficacy and tolerability of lenalidomide has been conducted and it compared lenalidomide 5 versus 25 mg/day, administered during 6 months, or until progression, in 60 patients, without hormonal therapy, after PSA relapse. Main toxicity was neutropenia, thrombotic events, asthenia and rash, with more grades 3–4 events in the 25 mg dose arm. Despite higher toxicity, PSA decline curve was favourable to patients receiving the 25 mg/day dose. The first results of a phase II trial combining bevacizumab, lenalidomide, docetaxel and prednisone in CRPC patients were presented at ASCO meeting 2011.

Among 24 patients who had completed four or more cycles, 22 patients had a >50 % PSA decline, and 20 patients had >75 % PSA decline, 14 patients, with measurable disease, showed 2 RC, 9 PR and 3 SD (overall response rate of 78.6 %). Therefore this combination seems to be associated with a high response rate with manageable toxicity. A phase III trial comparing different doses of lenalidomide combined with docetaxel-prednisone versus placebo is, however, currently underway (Merino et al. 2011).

Although one might conclude from these studies that antiangiogenic therapies are ineffective in mCRPC, we believe these negative data highlight an important biologic principle in prostate cancer angiogenesis that should inform the design of future trials (Antonarakis and Armstrong 2011). Specifically, the bone marrow microenvironment contains multiple proangiogenic factors in addition to VEGF including PDGF, basic fibroblast growth factor (bFGF), interleukin 8, and other soluble cytokines. This multiplicity of angiogenic pathways creates “redundancy” and the potential for “tumor escape” from antiangiogenic therapies and suggests that blocking multiple pathways simultaneously, rather than VEGF alone, may be necessary to effectively block angiogenesis in mCRPC. In support of this, our experience with clinical trials suggests that blocking PDGF and VEGF simultaneously (with sunitinib) is more potent in eliciting PSA responses in patients with mCRPC than blocking either VEGF alone (with bevacizumab) or PDGF alone (with imatinib) (Chi et al. 2005). Reflecting these data, studies are currently underway using tyrosine kinase inhibitors that target multiple angiogenic pathways (e.g., TKI258, which potently blocks VEGF, PDGF, and bFGF) (ClinicalTrials.gov identifier: NCT00831792), or alternatively, combine agents that block angiogenesis through different mechanisms (e.g., combining bevacizumab plus lenalidomide). In addition, in a recent phase I/II study combining sunitinib and docetaxel for the treatment of mCRPC in the frontline setting, patients demonstrated reductions in both PSA levels and tumor burden that were more substantial than a historical cohort of patients receiving docetaxel alone (Fig. 14.1) (Sowery et al. 2008).

Fig. 14.1
figure 1

Therapies targeting angiogenic pathway. Vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1, VEGFR-2 and VEGFR-3) provide for new vessel formation and their maintenance. VEGF expression is markedly higher in prostate cancer specimens compared to non-neoplastic prostatic tissue controls and plasma VEGF levels are significantly higher with metastatic versus localized disease. Bevacizumab is a recombinant humanized IgG1 monoclonal antibody with high affinity and specificity for all isoforms of VEGF-A. It binds to soluble VEGF-A limiting ligand binding to EC receptors VEGFR-1 and VEGFR-2 and blocking the transduction of proangiogenic intracellular signals. Sorafenib, sunitinib and Cediranib are multitargeted receptor tyrosine kinase inhibitors (TKI), that exerts their antiangiogenic effect targeting, respectively, RAF kinase, VEGFR-2 and platelet-derived growth factor receptor (PDGFR-β), the three VEGFR isoforms, PDGFβ and KIT, and VEGFR 1 and 2

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The observation that both bevacizumab and sunitinib have shown prolongation of progression-free survival without differences in overall survival also raises the possibility that sustained suppression of angiogenesis is required to affect overall survival (Antonarakis and Armstrong 2011; Dayyani et al. 2011). Enhanced tumor growth following cessation of antiangiogenic therapy has been described, a “rebound” phenomenon that could influence overall survival (Chi et al. 2009). To address these limitations, it may be necessary to continue antiangiogenic therapy beyond standard definitions of disease progression to observe a beneficial impact on overall survival.

There are many question to be answered to optimize antiangiogenic therapy for advanced prostate cancers.

  • The role of several angiogenic regulator factors is still poor understood. As an example, we currently know that the prostate-specific membrane antigen (PSMA) expression in tumor-associated neovasculature is necessary for angiogenesis and endothelial cell invasion, but we are unaware of its real role in angiogenesis (Gordon et al. 2008).

  • As well, VEGF activation is probably mediated by other still unknown transcription factors such as the Activator protein 1 (AP-1) transcription factor complex (Shih and Claffey 1998).

  • Further studies will also address the predictive role of expression of HIF-1 alpha, VEGF, and other angiogenic growth factors in patients treated with radiotherapy alone. These patients, in fact, lack the beneficial effect on tumor vascularization exerted by a neoadjuvant androgen deprivation. Therefore, the angiogenic markers may be even more important in this subgroup of patients (Vergis et al. 2008).

Anyhow, it is a matter of fact that almost all the key regulators of angiogenesis are upregulated in prostate cancer, particularly in the castration-resistant setting, and this undoubtedly has a great relevance for the gain of prostate cancer aggressiveness. This strongly stimulates the search for new reliable marks for effectively targeting prostate cancer angiogenesis.