2.4.1. Angiogenesis as a hallmark of cancer
The hallmarks of cancer define distinctive and complementary capabilities that allow tumors to grow and disseminate. One of those capacities is the induction of angiogenesis. This process specifically refers to the sprouting of new blood vessels from pre-existing ones, involving proliferation of endothelial cells and migration towards pro-angiogenic molecules. The expansion of the existing vasculature also relies on the accumulation of circulating en-dothelial progenitor cells. The latter are immature endothelial cells, typically arising in the bone marrow, with the capacity to extravasate in response to pro-angiogenic factors and promote new vessel formation known as vasculogenesis. This process also takes place in the tumor microenvironment; however, it is generally associated with embryogenesis and development and involves the birth of new endothelial cells and their assembly into tubes in addition to the sprouting. Following this morphogenesis, the normal vasculature results in a quiescent action, becoming in the adult only an active process in wound healing events and in female reproductive cycling, but only transiently.
The tumor and its microenvironment display a completely different scenario, allowing pro-inflammatory molecules to switch on the angiogenic process enabling the tumor to grow, persist and disseminate. The tumor-associated angiogenesis was previously considered to be important in growing macroscopic tumors; however, the clinical evidence show that it directly contributes to the microscopic premalignant phase of neoplastic progression, further securing its position as an integral hallmark of cancer. This angiogenic switch is governed by angiogenic regulators that bind to stimulatory or inhibitory cell-surface receptors displayed by vascular endothelial cells. The well-known inducers of angiogenesis include among others: VEGF-A, TGFp and IL8; while inhibitors include: thrombospon-din-1 (TSP-1) and angiostatin, among others. In tumors, these molecules support the rapid division of tumor cells . VEGF signaling occurs via three main subtypes of receptor tyrosine kinases known as VEGFR1, VEGFR2 and VEGFR3. Its expression can be upregulat-ed both by hypoxia and by oncogene signaling [99, 100]. Additionally, VEGF ligands can be sequestered in the ECM in latent forms that can then be activated by ECM-degrading proteases such as MMP9. Also the fibroblast growth factor (FGF) family is capable of activating VEGF and has been implicated in sustaining tumor angiogenesis. TSP-1 emerges as a counterpart of the angiogenic process, that when activated suppresses proangiogenic stimuli . Of note, Ras and Myc, dominant oncogenes can also upregulate angiogenic factors in the tumoral microenvironment, and these signals can also be produced indirectly by immune inflammatory cells.
It is of particular interest the fact that angiogenesis inhibitors, such as TSP-1, angiostatin and endostatin offer natural barriers to tumor angiogenesis. This was described by Ribatti et al. , followed by several studies reporting other endogenous inhibitory agents. Most of these molecules appear to derive from proteolytic cleavage of structural proteins that are not angiogenic regulators per se, and some can be detected in normal mice and human plasma. These agents serve under normal circumstances as physiologic rheostats modulating angio-genesis during tissue remodeling and wound healing but may also act as intrinsic barriers to the sustained angiogenesis in emerging neoplasias.
How do these counterpart molecules behave in the tumoral process? How can we decipher the cross talk of this aberrant mix of proangiogenic signals? A massive amount of information describes the features of a cancer cell. However, it is wise to acknowledge the differen tial concepts of causes, oncogenic events, signal transduction programs, and hallmarks to show that there is a complexity under this network of interrelations that dynamically changes in different cells, between cells, and most importantly at different times in any given cell. Cancer is an evolving, heterogeneous system, hence the intricacy of the forming vas-culature supporting tumor growth and progression.
While sprouting angiogenesis requires VEGF for endothelial cells to proliferate, migrate and maturate into new vessels, in the absence of this factor, the blood vessels split into new vessels without the need of endothelial cell proliferation. This phenomena is termed intussusception and has been demonstrated in various tumors . Intussusception cannot be stopped by anti-VEGF strategies.
Intussusceptive microvascular growth refers to vessel network formation by insertion of connective tissue columns, called tissue pillars, into the vessel lumen and to the subsequent growth of these pillars, resulting in the sub-division of the vessel lumen. Intussusception is observed in a variety of normal and malignant tissues. It is faster and more inexpensive than sprouting, occurring within hours or even minutes and besides its autonomy from endothelial cell proliferation, it also becomes independent from basement membrane degradation, or even invasion of the connective tissue. However, intussuscep-tive microvascular growth displays a limiting factor: it can only work on existing vessel networks. Therefore intussusceptive microvascular growth has the ability to increase the complexity and density of the tumor microvessel mesh already built by sprouting. Although the molecular networks underlying this vascularization mechanism are poorly understood, the role of some local stimuli, such as intravascular shear stress, may induce a cascade of physiological or pathological reactions in endothelial cells, such as new capillary development by tissue pillar formation .
The absence of intense endothelial cell proliferation in intussusceptive microvascular growth implies that neovascularization by this mechanism would be resistant to angiosup-pressive treatment in itself. Clinically, accumulation of tumor blood vessels by intussuscep-tive vessel growth is associated with a poor outcome for various types of cancers .
Until recently, vascularization of malignant tumors was considered the exclusive result of directed capillary ingrowth (endothelial sprouting). However, recent advances have been made in identifying the processes involved in angiogenesis and vascular remodeling. Consequently, the simplistic model of an invading capillary sprout has been deemed insufficient to describe the entire spectrum of morphogenic and molecular events required to form a ne-ovascular network. Cancer tissue can acquire its vasculature by co-option of pre-existing vessels, intussusceptive microvascular growth, postnatal vasculogenesis, glomeruloid angiogenesis, or vasculogenic mimicry [103, 105].
Before discussing the different ways a tumor is vascularized, we should highlight that these mechanisms may not be mutually exclusive; the literature has shown that in most cases there is a cross-talk between these systems, participating in conjunction in physiological as well as in pathological angiogenesis. Although the various types of cancer vascularization may share similar molecular signaling cascades and may be controlled partly by almost identical regulatory factors, a significant variety of differences also prevail.
It is widely accepted that the primary tumors and metastases have an initial avascular growth stage and then the angiogenic switch is turned on to support the exponential tumor growth. Tumor-induced angiogenesis and tumor cell vessel interactions are one of the most important events during all the stages of tumor development. However, it is not fully understood what is exactly happening before or during the initiation of vascularization of the primary tumor and the micrometastasis. In the beginning malignant cells may associate with and grow preferentially along pre-existing microvessels, prior to building their own vasculature. This process is called vessel co-option and was first proposed by Holash et al. . Although at first, it is limited to the early stages of human tumorigenesis, morphological evidence suggests that co-option of pre-existing blood vessels might persist during the entire period of primary or metastatic tumor growth. During solid tumor growth, no signs of directed vessel ingrowth can be appreciated; instead, these tumors decide to develop by co-opting the massive vascular plexus present in the peritumoral connective tissue. Several controversies have been raised regarding how tumors progress, whether microtumors may initiate growth by exploiting pre-existing vessels without inducing angiogenesis or initiating through the induction of angiogenic sprouts from host vessels . These discrepancies may have aroused given the differences in vascular niches in applied experimental models. Although unresolved from a mechanistic point of view, this uncertainty may raise important challenges when outlining a rationale for therapeutic strategies. This implies that, whereas compounds may be efficient inhibitors of angiogenesis and tumor growth in angio-genesis-dependent tumors (such as subcutaneous tumor xenografts), their effects may be limited in tumors growing in tissues with an intrinsic vascular density that allows for co-option by infiltrative tumors or other forms of neo-vasculature.
Based on this knowledge, new ways to inhibit the various vascular modalities have been developed in the past decade. When applying these targeted therapies, there are several aspects to take into consideration: the stage of tumor progression, the type of vascularization of the cancerous tissue and the molecular signaling networks behind the vascularization process.
What are the key aspects in determining the vascularization patterns of tumors? First, the local microenvironment, important during tumor initiation. Second, the cell number, subsidizing microtumors ability of inducing angiogenesis. Moreover, to trigger exponential growth, tumors must depend on vascularization through angiogenesis, which is much more powerful than vessel co-option to increase the tumoral mass and to acquire nutrition and oxygen from the host circulation system. If possible, tumors will prefer this kind of vascula-rization pattern. Alternatively, another choice is the strategy of co-opting host vessels in order for tumor cells to survive when they cannot acquire enough support from its niche and have no capacity to establish intrinsic vessels through angiogenesis. This is consistent with the observations that anti-angiogenic therapies result in an increase of vascular co-option
. Third, the co-option and migration along host vessels will be inhibited once angiogen-ic sprouts begin to be induced.
Of note in liver metastases of human colorectal carcinomas, different growth patterns can be observed, depending on the degree of differentiation. These liver metastases represent a truly heterogeneous group and their growth patterns (replacement, pushing and desmoplastic) predict the fraction of immature blood vessels, the fraction of proliferating endothelial cells and the fraction of apoptotic tumor cells. The replacement growth pattern expands mainly by co-opting the stroma with the sinusoidal blood vessels of the liver .
The use of anti-vascular endothelial growth factor antibodies have been used for the abrogation of angiogenesis and growth of human prostate carcinoma microtumors and even metastasis in orthotopic prostate cancer xenografts. Although up to date there are no reports suggesting that vessel co-option is also an alternative route for growth and dissemination of prostate tumors, the contribution of this vascular route to prostate tumorigenesis needs further exploration; specifically, the involvement of this survival tool for growth of microtumors [110, 111].
Many studies have reported the close association between host vessels and extravasated cells during the onset of metastases. The co-opting manner makes these tumoral cells cover vessel surface area as much as possible and obtain the necessary support from host, such as nutrients or oxygen, with remarkable vessel-like pseudopodia. As Weinberg articulated for this kind of behaviour "tumor cells require effective interactions with the vasculature in order to acquire nutrients and to shed metabolic waste products and carbon dioxide In some normal tissues with an especially high metabolic activity, most cells enjoy direct contact with at least one capillary. This intimate association means that their access to oxygen and critical nutrients not dependent on the diffusion of these molecules over large distances and through densely packed cell layers" .
The tumoral vascular picture clearly displays differential contributions of vessel co-option and angiogenesis at the earliest stage of tumor initiation and metastasis. While angiogenesis appears as a key player for tumor exponential growth, the strategy of co-opting host vessels seems indispensable for cancer cell survival. Future anti-vascular therapies should seriously take into consideration the alternative ways in which a tumor disseminate and evades conventional anti-angiogenic treatments.
How can we distinguish normal angiogenesis from tumor-associated angiogenesis? Tumor neovasculature is marked by precocious capillary sprouting, convoluted and excessive vessel branching, distorted and enlarged vessels, erratic blood flow, leakiness leading to blood lakes, and distorted levels of endothelial cell proliferation and apoptosis . Also, certain types of cancer cells have the capacity to mimic the activities of endothelial cells and to participate in processes that involve the formation of a fluid-conducting, matrix-rich meshwork, metamorphosing into vessels that either carry blood or connect to the host's blood supply. This new mechanism, by which some aggressive tumors may acquire a blood supply, was first described by Maniotis and coworkers  and was termed 'vasculogenic mimicry'. However, it cannot be considered a vasculogenic event as true vasculogenesis involves de novo formation of endothelial cell-lined vessels. Since its discovery, vasculogenic mimicry has been catalogued in several types of tumors. How does vasculogenic mimicry contribute to tumor growth and progression, and can it be targeted by therapeutic agents?
Several interpretations of vasculogenic mimicry have evolved since tumor angiogenesis was recognized as not the only mechanism of blood supply for tumor microcirculation. Vasculo-genic mimicry describes the ability of aggressive tumoral cells to express endothelium-asso-ciated genes and to form ECM-rich vasculogenic-like networks in three-dimensional (3D) cultures. These new vessels have no endothelial lining and are mainly composed of basement membrane-like material. The formation of these networks, seem to mimic the embryonic development of vasculogenic meshes and they were associated with the distinctly patterned ECM-rich networks that are observed in aggressive tumors. Since its discovery, vasculogenic mimicry has been described in several kinds of tumors, including melanoma, synovial sarcoma, rhabdomyosarcoma, osteosarcoma, breast carcinoma and ovarian carcinoma. Most of these studies correlate the aggressiveness of the tumor with angiogenesis or vasculogenic mimicry proliferation . But how do they form and what is their contribution to tumorigenesis?
In the beginning, researchers observed in xenograft models and human biopsies, patterned loops and arcs that confined spheroidal clusters of tumoral cells. These loops and arcs formed networks that were lined with cancer cells and contained laminin and other components of the ECM yet not explored. Studies of tumor-tissue sections showed that the spheroidal tumor clusters contained either small, channel-like spaces between them, or seemed to be partially or totally juxtaposed by ECM. Some of these channel-like spaces were originally defined as 'vascular channels', because they were found to contain erythrocytes and plasma and were thought to provide a perfusion mechanism and a dissemination path within the tumor that might work independently or together with angiogenesis or vessel co-option.
Blood lakes within the tumor are another physiological phenomena that also draw attention. These are large collections of extravascular erythrocytes lining tumor spaces or channels. As hemorrhage is a manifestation of the defective endothelial barrier function in tumors the reason as why some tumors are bloodier than others, might rely on the balance between er-ythrocyte extravasation and the vessel wall stability. Rapid endothelial cell proliferation and defective pericyte coverage might contribute to the instability of tumor vessel walls leading to this hemorrhage. Pericytes are supporting cells that are closely apposed to the outer surfaces of the endothelial tubes in normal tissue vasculature, providing mechanical and physiologic support to the endothelial cells and have been associated with the maintenance of a functional neo-vasculature of most if not all tumors .
The literature on vasculogenic mimicry in prostate cancer is scarce, although therapeutic implications of it have been described in aggressive prostate cancer in vitro . The prognostic value of vasculogenic mimicry remains debatable as there is at least one study showing that there is no significant correlation between vasculogenic mimicry channels and histolog-ical grading of prostate cancer .
Interestingly, Liu et al.  looked at this correlation in human tissue samples to determine clinical pathology, prognosis and a possible molecular mechanism. They statistically correlated histological with clinicopathological data from prostate carcinoma cases confirming that vasculogenic mimicry was more often seen in those patients with seminal vesicle invasion, lymph node metastasis, distant metastasis tissues or shorter PSA doubling time (PSADT), all important clinical prognostic factors of prostate cancer. They concluded that vasculogenic mimicry mainly exists in the high-risk prostate cancer patients and is a new independent marker of poor prognosis of the disease. Though more studies with larger sample sizes are needed to further confirm the correlation of vasculogenic mimicry and prostate cancer prognosis, these results might explain why some anti-angiogenesis treatments remain clinically less effective.
The identification of molecules that are uniquely expressed on the surface of endothelial cells of tumor vessels has been a holy grail of vascular biology. Such molecules could serve as therapeutical targets. Although there is no molecule truly associated to tumor vessels, several show higher expression in tumors. Among those relevant in prostate cancer we find: endoglin (CD105), VEGF/VEGFR-2 complexes, thrombospondin-1 receptor (CD36), Thy-1 cell surface antigen (Thy-1), phosphatidylserine, prostate-specific membrane antigen (PSMA), MMP, Her2/Neu and multiple tumor endothelial markers. The absence of absolute specificity of these molecules for tumor vessels drives the search for better targets . Of note, Her2/Neu plays an important role in the spreading of prostate carcinomas to the bone and its high expression is associated with a poorer prognosis in patients with bone metastases. The Her2/Neu receptor is part of a molecular signaling cascade that involves Akt and MMP-9 activation, enabling the cancer cell to penetrate the matrix and facilitating angiogenesis.
It is wise to recognize the lead role of MMP in facilitating the invasiveness of prostate cancer. These molecules are important in the degradation of the ECM, allowing tumoral cells to metastasize to distant sites throughout the body. This protease activity, not only allows for cell migration, but also facilitate angiogenesis, providing the tumor with nutrition and further proliferation . Of note, MMP-2 plays an important role in the preliminary stages of the vasculogenic mimicry genesis, degrading collagen IV. Reports showed that human prostate carcinoma samples positive for vasculogenic mimicry had a significantly higher MMP-2 expression levels compared to vasculogenic mimicry-negative patients. Metastat, an inhibitor of MMP, decreased the formation of vasculogenic mimicry networks in aggressive prostate tumors. However, further studies are needed to elucidate the mechanism of formation of vasculogenic mimicry in detail .
In bone metastases, the prostate metastatic tissue might allow for angiogenesis via the MMP9 derived from osteoclasts. Interesting, some MMP have a higher expression with higher Gleason's scores. This fact has led to the revamping of the MMP as possible prognostic factors and even more, as valid candidates for therapy. However, the MMP field is at a crossroad; in the last few years, accumulating evidence from experimental models of cancer, knockout mice and proteomics studies has challenged our views on how MMP function in the tumoral process. This challenge has been compounded by the fact that the clinical trials with MMP inhibitors failed to show therapeutic efficacy in cancer patients. MMPs have a vast repertoire of substrates not limited to the ECM components, and multiple proteins can be potentially targeted by MMPs and may be important for the anti-tumor activity of the host. This may partly explain why broad-spectrum synthetic MMP inhibitors failed to show clinical efficacy.
The MMP picture is not simple and reveals a complex contribution to cancer progression, putting aside the long-held view of MMP as a family that promotes cancer metastasis. Today, the evidence shows that members of the MMP family may promote or inhibit cancer development. Moreover, an individual MMP may act positively or negatively on tumor progression depending on other factors, on the tumor stage, tumor site (primary, metastasis), enzyme localization (tumor vs. stromal) and substrate profile . In the -omics era, the identification of the substrates targeted by MMP in biological samples, known as degrado-mics, promises to become an important tool for defining the role of MMP in cancer. Establishing correlations, particularly in advanced prostate carcinomas, may assist in better patient stratification.
In fact, more questions than answers have been raised about the relevance of the in vivo studies on tumor vasculature. Is there a morphological and functional connection between prostate tumor-cell-lined networks and endothelium-lined vasculature? Is it possible for aggressive prostate cancer cells to form functional vessels when placed in an ischaemic, non-tumor microenvironment? What is the potential relevance of a 'plastic' tumor-cell phenotype, and how can we identify and target tumor cells that can masquerade as other cell types? Many of the biological properties that are relevant to embryogenesis are also important for tumor growth. For example, during embryonic development, the formation of primary vascular networks occurs by the process of vasculogenesis (the differentiation of mesodermal progenitor cells (angioblasts and hemangioblasts) to endothelial cells) and their organization into a primitive network . The remodeling of the vasculogenic network into a more refined microvasculature occurs through angiogenesis in the same way as tumors require a blood supply for growth and also use the blood supply for metastatic dissemination .
Cells capable of vasculogenic mimicry display a high degree of plasticity, causing them to resemble dedifferentiated cell types. A stem cell is considered the most dedifferentiated cell, holding the capacity to generate various novel cell types. However, a new concept comes into the picture, the cancer stem cells (CSCs). These cells hold the capacity to self-renew, differentiate and proliferate indefinitely, being the latter a key event in tumor growth. Tumoral vasculogenic mimicry is characterized by an undifferentiated molecular signature together with embryonic-like differentiation plasticity implying a link between cancer stem cells and aggressive tumor cells capable of vasculogenic mimicry. Moreover, these two cell types share the potentiality of unlimited proliferation capacity, cellular plasticity and the expression of a gene signature responsible of maintaining pluripotency.
Among the signaling molecules known to influence stem cell renewal and differentiation in aggressive forms of prostate cancer, we find: Wnt, Src, BMP (bone morphogenic proteins) and TGFp . Other transcription factors are also involved in bone metastasis. HIF1a in tumor cells, inhibits osteoblasts differentiation, induces osteoclasts differentiation and promotes tumor growth. Hypoxia and TGFp signaling in parallel drive the development of tumor bone metastases and regulate a common set of tumor genes stimulating the production of VEGF and CXCR4 in both tumor cells and bone microenvironment to enhance angio-genesis and tumor homing. VEGF, a target gene of Runx2, facilitates tumor growth and both the osteolytic and the osteoblastic disease [123, 124]. Additionally, prostate cancer cell lines express mediators of tumor growth and bone destruction, among them IL8, IL6 and PTHrP. Runx2 is also a key regulator of metastasis related genes and its presence in the primary tumor could be critical for the diagnosis of prostate cancer bone metastasis .
The Notch signaling pathway is now recognized as an important player in tumor angiogen-esis. Two key Notch ligands have been implicated in this process, Delta-like 4 (Dll4) and Jag-ged1. Notch appears to be very attractive because specifically, bone metastases from prostate cancer patients expressed Notch-1 protein in the osteoblastic lesions. Correspondingly, Notch ligand Jagged-1 was found to be highly-expressed in metastatic prostate cancer compared to localized disease or benign prostate tissues, and high Jagged-1 expression in a subset of clinically localized tumors was found to be significantly associated with tumor recurrence . Although the molecular mechanism of Notch signaling is not completely understood, silencing of Notch-1 inhibits MMP9, uPA and VEGF expression, given support to the effect of Notch in invasion [126, 127]. Moreover, Wang et al  recently proposed a down-regulated signaling cascade downstream of Notch-1, with reduced Akt and mTOR phosphorylation and inactivated NF-kB signaling. The interplay between these pathways provides a balance between self-renewal and differentiation. Dll4 expression activates Notch resulting in restriction of new sprout development. In agreement with this activity, inhibition of Dll4-mediated Notch signaling in tumors results in hyper sprouting of nonfunctional vasculature . This Dll4 inhibition may paradoxically lead to increased angiogenesis but poor tumor growth because the newly growing vessels are not functional. In contrast, Jag-ged1 has been described as a Notch ligand expressed in tumor cells that may influence tumor angiogenesis by activating Notch on tumor endothelium. Of note, Notch activation is also critical for the maintenance of stem cell self-renewal potency in several stem cell microenvironments. These results indicate that Notch signaling can have diverse signaling outcomes dependent on the cellular niche, as it is able to induce (endothelial) differentiation in some cases, while promoting self-renewal potency in others .
TGFp signaling also draws our attention given that it is a key molecule in the maintenance of an undifferentiated state in human embryonic stem cells. Various components of the TGFp signaling cascade are highly expressed in stem cells, including Nodal and its regulators Cripto and LEFTY1/2 [101,102]. However little is known about signaling cascades governing the pluripotent state . Taken together multiple stimuli provided by prostate tumors and their effective microenvironment can trigger differential signaling cascades that in turn will define the fate of the host. Thus a variety of therapeutic venues may have to coexist in order to be translated into clinical utility.
Undoubtfully, there are more questions than answers at this time regarding the functional significance of vasculogenic networks and vascular marker expression by prostate cancer cells. If tumor vasculogenesis can be demonstrated in experimental models, does it occur concomitantly with angiogenesis or as a remodeling of angiogenesis in aggressive tumors? Is vessel co-option involved? Is tumor cell vasculogenesis an alternative angiogenic switch in aggressive tumors? Regardless of the terms employed to describe the expression and mimicry of vascular-like gene by aggressive prostate cancer tumor cells, this area of research is worthy of analysis. It is wise to consider that in addition to the current anti-vascular treatments, the novel therapeutic approaches against tumor vasculature must be harmonized with the stage of tumor progression and with the molecular mechanism responsible for the angiogenic phenotype.
In our perspective the challenge relies in combining the anti-vascular strategies with the existing therapeutic regimes. The rational application of antivascular agents must be tagged along with the notion that these therapies must be individually tailored for the different types of cancer cells. The clinical management of prostate cancer would benefit greatly from the better understanding of the diverse vascularization mechanisms helping to fine-tune these novel anti-cancer strategies.
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