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Writer's pictureAnhLe

I found the literature review I did in my final undergrad year...

Title: Hypoxia, angiogenesis and cancer metastasis:

When it gets stuffy, you need to get out!


Student: Anh Hoang Le

Supervisor: Professor Kate Nobes

Literature Project

Year 2015-2016

“This project is my own work except where indicated. All text, figures, tables, data or results which are not my own work are indicated and the sources acknowledged”

Word count: 5484


Abstract

Metastasis is responsible for ninety percent of deaths by cancer every year. There are many factors that contribute to the spread of cancer cells. This can be intrinsic mutations such as those which lead to the loss of cellular adhesions or overexpression of metalloproteases, or external such as the presence or absence of blood vessels in tumours. Many important discoveries were made in studying these changes, but this review will only focus on the biology of solid tumours and the two important aspects affecting cancer spread. These include the hypoxic microenvironment, with an emphasis on the Hypoxia-inducible factor 1, and the consequences of hypoxia on the regulation of angiogenesis – the process of forming new blood vessels – through vascular endothelial growth factor signalling. At the end of the review, we will also discuss how the knowledge of the tumour microenvironment is applied in cancer treatments, and the challenges that we are currently facing and possible solutions.

I. Introduction

The marking point for when a tumour becomes malignant is when the dissemination process occurs, or in another word, the tumour starts to metastasize. Ninety percent of deaths [51] from cancer are due to this process. However, the question of how and why do cancers spread has bugged researchers for decades. Unfortunately, there is no single answer to this question. In order for a cancer cell to migrate from its primary tumour to a new location, many changes from both the inside as well as the outside environment need to take place. Changes in adhesion molecules such as E-cadherins [15, 25, 40] and integrins [8] will make the cells more mobile and ready to detach from the tumour mass. Changes in the organisation of the cytoskeleton, the regulation of many actin nucleating factors [44, 48, 78] and expression of surface-bound metalloproteases [19] give cancer cells the ability to crawl, to migrate and to digest through barriers. Along with this is the acquirement of resistance to programmed cell death [28, 37] by either ignoring death signals or blocking pro-apoptotic proteins such as Bad or Bax.

For many years, scientists thought that a tumour mass was a homogenous structure with each cell experiencing the same environmental conditions. However, it is because of this misunderstanding, many observations and phenomena in tumour biology could not be explained, such as the presence and interactions of tumour-associated leukocytes with cancer cells, or the different behaviours and histology of cells within a tumour (internal heterogeneity) [7, 46]. It is only recently that the study of the tumour microenvironment has become so important in understanding these events. Many factors have been shown to favour the development of many evolutionary advantages for cancer such as the loss of heterozygosity, or gain-of-function mutations. For solid tumours, however, the hypoxic response [6] is considered to be one of the major contributors to the transformation from a benign to a malignant state.

Tumour hypoxia is a state where the partial pressure of oxygen within the core of the tumour decreases below the physiological pressure measured in the normal surrounding tissue, which in turn depends on the type of tissue that the tumour originates from. The reaction of tumour cells to this condition is potent enough to make them become more aggressive and invasive. The main protein that is involved in this response is the Hypoxia-inducible factor (HIF). The HIF protein family in human comprises of many different members including HIF-1, 2 and 3 with each has two isoforms  and , but in this review, we will only focus on HIF-1. It has long been well-established on the positive effect of HIF-1 on the expression of vascular endothelial growth factor (VEGF). This is no coincidence that the upregulation of VEGF is one of the hallmarks of cancer, especially when this is the sign when the tumour size has surpassed the diffusion limit of oxygen and nutrients, thus favours the dissemination of cancer cells and leaves patients with a very poor prognosis. Hence, a major part of our discussion will emphasise on the HIF-1-dependent VEGF expression and the regulation of angiogenesis. We will also mention other HIF-1-independent angiogenesis mechanisms in brief, which also play an important role. Treatments derived from the understanding of tumour hypoxia are under intense research, with some of the major success including the using of monoclonal antibodies. Like any other cancer therapies, difficulties arise when it comes to human trials, with some treatments showed no outcome while others showed severe side effects. These problems along with possible solutions will be discussed in detail near the end.

II. Main text

1. The hypoxic microenvironment of solid tumours

1.1. Probing the hypoxia

The idea of a microenvironment within tumours has been around for many years. Cancer biologists believe that the main factor that contributes to the differences between an internal environment of a tumour and the surrounding is the oxygen level. Biochemical analyses identify HIF-1 as an upregulated protein under hypoxic conditions from tumour biopsies, but a more direct proof on the oxygen level is still needed. Many different techniques have been utilised to “peek” inside tumours, which includes electron paramagnetic resonance imaging, fluorescence redox imaging or diffuse optical spectroscopy (DOS) [80]. Figure 1 describes DOS as an example.




Figure 1. a. The structure of the side-firing fibre optic probe which can be attached to b. the skin of the animal and measure the hypoxic level of the tumour spectroscopically and transmit the signal to the main DOS instrument. Adapted and modified from [80].


Indeed, it was found that the partial pressure of oxygen at the core of many solid tumours is only about 10mmHg, compared to normal tissues which are usually about 40 to 65mmHg [3]. Combining this with the current understanding of cancer biology, it is not so surprising that this is the case. Cancer cells express a faster rate of proliferation and division compared to normal cells. On one hand, this gives them the advantage to quickly overcome normal cells in term of spatial occupation, while on the other hand, this poses a nourishment problem, particularly for solid tumours. As cells divide, the mass of the tumour increases, the demand for oxygen exceeds the supply, and because the diffusion limit of oxygen is only about 100 to 200um [83], these lead to some areas of the tumour experiencing less oxygen than others and becoming hypoxic. Even regions that are close to blood vessels can also be subjected to hypoxia; this is due to the fluctuation of the blood supply [22], which comes from the malfunctioning tumour vessels which will be discussed later.

This direct evidence of tumour hypoxia has set a firm and fundamental foundation for understanding how tumour cells behave and the resulting consequences that we have observed. Development of probing methods also opens a way of monitoring the effects of external factors on the tumour, such as those of pharmaceutical agents.


1.2. The biology of Hypoxia-inducible factor 1 (HIF-1)

HIFs are a group of transcription factors that are involved in many fundamental cellular processes. Different HIF subunits combine to form different types of HIF proteins. This family of proteins controls cellular responses to oxygen, including developmental processes such as of pancreatic beta cells [34] or angiogenesis [43, 52, 84]. HIF-1 is a heterodimeric protein composed of two subunits HIF-1a and HIF-1b. HIF-1b is constitutively expressed, independent of the oxygen level while the expression of HIF-1a is tightly regulated by various enzymes and factors [35]. The general mechanism of HIF-1 regulation is described in Figure 2.




Figure 2. General regulation mechanism of HIF1

Red arrows represent inhibition, green arrow represents activation. PHD: Prolyl Hydroxylase, FIH-1: Factor Inhibiting HIF-1, NF-kB: Nuclear Factor k B, HRE: Hypoxic Response Element, VEGF: Vascular Endothelial Growth Factor.


In normoxic conditions, HIF-1a is suppressed by two different pathways. A group of enzymes called prolyl hydroxylases (PHDs) uses O2 to hydroxylate HIF-1a at two different Prolyl residues. Then, von-Hippel Lindau protein (VHL) can ubiquitinate HIF-1a and target it for proteasomal degradation. In addition, the activity of HIF-1a is enhanced by the coactivator p300 and this interaction is inhibited by Factor inhibiting HIF-1 (FIH-1) [3]. At the transcription level, the expression of HIF-1a gene is directly monitored by the nuclear factor kappa B (NF-kB) [75]. However, at the hypoxic condition, such as within a tumour, neither PHD nor FIH-1 is active, while also there is an increase in the level of NF-kB in tumour cells, this shifts the equilibrium towards stabilising HIF-1a. HIF-1a dimerises with HIF-1b and translocates into the nucleus to switch on a wide range of different genes including VEGF via binding to its HRE sequences [3].

2. HIF-1, VEGF and tumour progression

2.1. VEGF and the regulation of angiogenesis

Before going any further into how and why angiogenesis is important in cancer metastasis, let’s briefly recap what happens during this process (Figure 3). The whole process is predominantly driven by the activity of the VEGF protein family, VEGF-A in particular [29, 56]. VEGF-A activates nitric oxide [38] production causing vasodilation and deposition of plasma proteins into the extracellular matrix (ECM) forming a “platform” in which integrin signalling will coordinate the movement of these endothelial cells. Activated VEGF receptors also activate Paxillin/FAK [38], which in turn suppresses the activity of Rho protein and increases focal adhesion turnover. This results in the destabilisation of the connection between endothelial cells and pericytes, priming for the blood vessel sprouting process. One of the endothelial cells will then be chosen as the tip cell. Neighbouring cells are then loosely moving along with the tip cell as well as dividing, forming a migration column. Finally, perivascular cells reattach and the basement membrane is reformed outside of the newly formed blood vessels [1, 10, 84].

This is a delicate process where each step is regulated precisely to result in functional blood vessels. One point that we will come back later in section 3.2. is that when tumours trigger angiogenesis, the resulting blood vessels are leaky and very poorly functioning. This suggests that some of the steps in the normal process are omitted. This is to speed up the rate of blood vessel formation to catch up with the high demand for nutrients of the tumour, and because of the natural leakiness of tumour blood vessels, malignant cells can easily enter the circulation to migrate to other locations in the body.




Figure 3. The cellular mechanism of angiogenesis

a. Stimuli trigger the dilation of blood vessels and loosening up cell-cell junctions, thus allow pericytes to detach and allow the endothelial cell underneath to move b. One of the cells will become the tip cell, which more or less displays physical characters like a neurone, with dendrite-like structures due to the activation of Rac and Cdc42 c. Neighbouring cells follow the tip cell and divide, forming a stalk or migration column, and finally d. the fusion between two vessels creates a new path for blood to flow through. Adapted from [1]


2.2. Hypoxia-induced HIF-1 – independent angiogenesis

The first important protein in HIF-1-independent angiogenesis is JUNB. JUNB is a subunit that makes up the heterodimeric transcription factor AP-1. The JunB gene is under the control of NF-kB. Inhibiting NF-kB using a super-suppressor IkB led to a significant decrease in the production of JunB mRNA. Explants that contain JUNB-/- cells develop a very poor vascular network and drop in VEGF mRNA [65], which suggests that JUNB is needed for the expression of VEGF gene.

The second protein is a small GTPase K-Ras. K-Ras is involved in controlling cell growth and proliferation. Many studies have shown the effect of K-Ras in stimulating angiogenesis in colon [62] and pancreatic cancer [45]. In colorectal cancer, knocking down HIF-1 showed a limited effect on angiogenesis (only 19-22% reduction in VEGF production), which suggested that HIF-1 might not play the main role here [50]. Deletion of the HRE on the VEGF promoter did not affect the expression of this gene, thus suggesting the HIF-1-independent characteristics of K-Ras [50]. It is proposed that K-Ras initiates the Ras/Raf/ERK signalling pathway [45], which eventually leads to the expression of a transcription factor ASP13 to activate VEGF gene. In fact, K-Ras still upregulates VEGF even under normoxia, but the expression level will significantly increase during oxygen-deprived condition [50].


2.3. Angiogenesis is a response of tumours to hypoxia

In normal physiology, the body maintains a balance between the stimulation and inhibition of angiogenesis. However, during the development of a solid tumour, this balance is tipped towards increasing blood vessel formation. This allows cells at the core of the tumour to get enough nutrients and oxygen for their survival, as well as to dispose of any metabolic waste like lactic acid, which can otherwise alter the pH of the surrounding environment. In response to the hypoxic condition, apart from the direct effect of oxygen on HIF hydroxylases, tumour cells respond to the release of the tumour necrosis factor a (TNFa) possibly from tumour-associated leukocytes [16]. TNFa then activates a kinase cascade, which phosphorylates the inhibitor of kappa B (IkB) and targets it for proteasomal degradation. NF-kB is now able to dimerise, translocate into the nucleus and activate the transcription of many genes such as HIF-1a gene. Knocking down NF-kB using siRNA led to a decrease of HIF-1a [36]. Additionally, the high level of HIF-1a also comes from the stabilisation effect of reactive oxygen species (ROS), resulting from the build-up of these free radicals by complex III in mitochondria [14]. Western blotting for HIF-1a in de-mitochondrial cells treated with hydrogen peroxide showed an increase in this protein level compared to untreated cells.

Elevation of HIF-1a level increases the expression of many HIF-targeted genes. Xenografts of the HIF-1a null cell line (c4) in mice were exposed to a hypoxic condition. The level of VEGF mRNA quantified by qPCR was significantly less than that from xenografts of wild type cell line. Staining for endothelial cell markers like PECAM revealed a reduction in the vascular network in tumours that lack HIF-1a genes. Further investigation showed a decrease in tumour mass formed by HIF-1a deficient embryonic stem cells [47].

It is worth mentioning that these results were not obtained from tumours that intrinsically formed in mice but were instead introduced artificially. Furthermore, xenograft tends to have little resemblance to the normal environment where tumours normally develop due to the lack of a vascular system, suitable ECM cell types and the natural heterogeneity. Nevertheless, we can still appreciate the significance of these data, which suggest the need for a good vascular network to maintain a tumour’s own integrity. Tumours that lack an adequate blood supply showed signs of necrosis. Indeed, the higher the level of HIF-1a, the more resistant and aggressive the tumour can get, the higher the chance for spreading, a point which we will come back in section 3.

At this point, you might have already noticed a very interesting, yet contradictory relationship between hypoxia, angiogenesis and the development of tumours. Hypoxia can drive tumour cell death, but at the same time, it has a positive influence that favours the aggressiveness of the tumour. Cancer cells in a malignant tumour are addicted to what is known as the Warburg effect [18, 39], in which the majority of ATP production comes from glycolysis and lactic fermentation, regardless of the presence or absence of oxygen. This poses a question on the real role of oxygen to cancer cells. Do tumours promote angiogenesis to crave for oxygen or just for other nutrients and that getting more oxygen is just an unavoidable consequence? Oxygen seems to cause more damage than benefit to cancer cells. Using oxygen can create ROS, which can damage the cell’s DNA, plus generating the set of enzymes needed for oxygen consumption is more expensive than using fermentation [2], so is the cost for tumours to induce angiogenesis and oxygen perfusion too high? This seemingly clear statement that angiogenesis is an adaptation mechanism of tumours to hypoxia is not so obvious after all.


3. Hypoxia and angiogenesis: The driver and the vehicle for cancer spreading

3.1. HIF-1a the master regulator of cancer metastasis in many cancers

Cancer metastasis is one of the hallmarks of malignant tumours. Once the tumour enters this process, the life expectancy for patients will drop dramatically. By definition, metastasis is a multistep process, in which cancer cells from a primary tumour move to distant locations within the body to form secondary tumours [8, 12, 32] (Figure 4).




Figure 4. General view of cancer metastasis

Cancer cells detach themselves from the highly vascularized tumour and enter a nearby blood vessel, in a process called intravasation. They travel in the circulation system until adhere to the blood vessel wall and undergo extravasation. The cells then divide and form secondary tumours. Adapted from [77]


The positive relationship between hypoxia and cancer has been known since the 60s. Nevertheless, the actual mechanism of how this happens is still an area of intense research. There is much evidence suggest that HIF-1a favours the spread of cancer. The first step in this whole process is the dissemination of cancer cells from the original tumour. Generally, normal cells are held tightly together by tight junctions, adherens junctions and desmosomes. Cancer cells, however, are much more disorganised. Cell junctions can still exist between cancer cells, however, because contact inhibition of growth is lost, connections can form at random angles. These connections can be lost and give rise to the detachment of cells away from the tumour mass.

E-cadherin, a classical cadherin molecule is well-known as a tumour suppressor protein [15], forms the major part of adherens junctions (Figure 5). Unsurprisingly, one of the main factors that contribute to the loss of this cell connection is HIF-1a. Experiments were done on renal cell carcinoma 4 (RCC4) [40]. These cells lack the protein VHL, thus allow them to naturally express a high level of HIF-1a. Immunoblotting confirms the absence of E-cadherins in these cells. Knocking-in VHL gene restored E-cadherin expression. Furthermore, if introducing the dominant-negative HIF-1a, a 9.8-fold upregulation of E-cadherins mRNA was detected. This strongly suggested that HIF-1 is a negative regulator of E-cadherins. It turns out that HIF-1 suppresses the transcription of the E-cadherin gene (CDH1) by binding to the gene’s HRE sequence. A qRT-PCR study showed a significant reduction in CDH1 mRNA level when HIF-1a was overexpressed. A recent study has identified a transcription factor called TWIST [79], which was shown to be positively regulated by HIF-1a. TWIST upregulates N-cadherin while diminishes E-cadherin to favour EMT and allow the detached cells to resist anoikis by activating Akt to suppress pro-apoptotic proteins like Bad and Bim. This is an evolutionary advantage because it allows cancer cells to move to another place, where more nutrients and oxygen are available.



Figure 5. Fluorescently labelled E-Cadherins of epithelial cells

As can be seen, E-Cadherins (green) decorate the periphery of these cells and connect these cells together in a sheet. In cancer, these interactions can be lost and are the first step of the epithelial-to-mesenchymal transition (EMT). The nuclei are stained with DAPI (blue). Adapted from Wikimedia Commons.


Not only the cancer cells are under the effect of hypoxia, but tumour-associated fibroblasts are also involved in favouring the migration of the cancer cells towards blood vessels. Gilkes, D. M. et al and other laboratories demonstrated the ECM remodelling effect of hypoxic fibroblasts in vitro [31]. Immunofluorescence microscopy showed a large deposition of collagen by these fibroblasts into the ECM and knocking down HIF-1a with shRNA effectively inhibits this secretion. The deposited collagen forms an ordered and almost resistant-free platform for cancer cells to slide on top.

Other studies also pointed out the activation of metalloproteases via uPA-uPAR signalling cascade in cancer cells under the hypoxic condition to allow them to degrade the native ECM and create a pathway to move through [60]. These secreted proteases also trigger the loosening of the endothelial cell junctions through interacting with PAR1 receptors, favours the intravasation process. Even though many of the experiments performed here were done on a 2D cell-free ECM system, the data were later validated in a 3D system using in vivo time-lapse imaging.

After detaching from the original tumour and entering blood vessels, cancer cells will then be carried by the bloodstream until they can dock onto the blood vessel wall and extravasate to form a new colony. Many studies have pointed out the effects of hypoxia still follow these cells throughout their journey. HIF-1a leads to the expression of L1 cell adhesion molecule (L1CAM) on [81] the cancer cells, which supports the extravasation step. Exposing L1CAM knock-in GFP labelled cells to 1% O2 for 48h then injecting them into the tail vein of mice and studying with fluorescence microscopy have shown a significantly larger proportion of extravasating cells compared to the L1CAM knockout cell line. Tail-vein injection experiments can pose some degree of uncertainty as we do not have a complete understanding of the interactions between the injected cancer cells and the environment that they travel through as well as the effect of different cell types onto the final behaviour of cancer. Nevertheless, the finding was further supported by Debbie Liao’s work, where she showed that transgenic mice – a more reliable model - that contain HIF-1a deficient mucoepidermoid carcinoma cells showed less pulmonary metastases than in wild-type mice [42]. Figure 6 summarises the complex interactions of HIF-1a in cancer cells.




Figure 6. The complex interconnected activities of Hypoxia and HIF-1 favours the progression and metastasis of malignant tumours. Green arrows: positive regulation, red blunt lines: negative regulation.


3.2. Tumour blood vessels are different from normal blood vessels

The process of angiogenesis described in 2.1 is a multistep process with the involvement of a plethora of hormones and chemokines. Every step in this process is tightly controlled to result in functional blood vessels. However, things are different with tumour angiogenesis, which was confirmed by many anatomical studies and functional assays. These blood vessels derive from pre-existing normal blood vessels but tend to be leakier, disorganised, irregularly shaped and can be blunt-ended [21, 49, 61]. This makes them very poorly functioning, even the tumour tissue that has the blood supply can still enter hypoxia, because of the fluctuation of the bloodstream passing through this type of blood vessels. This hints that the process of tumour angiogenesis occurs in a less well-coordinated manner, with fewer steps and less controlling factors.

The “porous” character of tumour blood vessels comes in part from the poor coverage by pericytes. In normal tissues, pericytes attach tightly on the periphery of blood vessels and provide an extra one-layer thick cellular sieve to control the movement of substances across the wall. These cells are lost or significantly reduced in number in tumour tissue. Neural cell adhesion molecule (NCAM) is responsible for the recruitment of pericytes. This molecule is activated as blood vessels branching out and especially after the fusion event occurs. In NCAM deficient tumours, observing pericytes using a-SMA showed frequent detachments from endothelial cells. Knocking-in NCAM in transplanted skin fibrosarcoma cells increased pericyte recruitment by 47% and coverage by 33% [77]. The actual mechanism that leads to the loss of NCAM has yet to be elucidated, but some suggest that this may be due to alternative mRNA splicing.

Leaky blood vessels lead to the efflux of blood plasma, which elevates the interstitial fluid pressure (IFP) inside the solid tumour. This physically exerts a force and increases the chance for cancer cells to undergo metastasis. Indeed, an inverse correlation exists between the percentage of pericyte-endothelial cell association and the number of metastases found in mice [17]. Abnormal blood vessels also increase hypoxia and the upregulation of many EMT genes (Figure 6). In addition, leukocytes can easily be recruited to the tumour by secreted chemokines from cancer cells. These leukocytes provide nutrients, growth factors and especially prostaglandin E2 – a signalling lipid molecule that promotes tumour development [16]. Overall, tumour blood vessels provide tumours not only with nutrients but also a perfect pathway for dissemination and proliferation, and that the combinatorial effect of angiogenesis and hypoxia contribute to the poor prognosis in patients.


4. Bridging the knowledge with treatment

Understanding the biology behind the tumour microenvironment and its effects on tumour development, especially in terms of angiogenesis has opened up a whole new area of therapeutic research.

4.1. Anti-HIF-1 therapy

Throughout our discussion, it is clear that HIF-1 plays important roles in the development of solid tumours, which include activation of angiogenesis, resistance to apoptosis, and favouring the metastasis process of cancer cells. Each step in the regulation of HIF-1a is a potential targeting point for therapy (Figure 7).



Figure 7. Each and every step of the regulation of HIF-1 can be a point of target for therapeutic agents. Some drugs target the transcription and translation process of HIF-1 such as Aminoflavone or mTOR inhibitors. However, many drugs act at the post-translation steps to affect the function and behaviour of the protein itself. Two prominent examples are Acriflavine and Anthracycline family. Adapted from [68]


Radiation therapy has been shown to induce HIF-1 expression [53, 67]. It oxidises water and oxygen to produce ROS, which will then translocate into the nucleus and induce DNA breaks. Paradoxically, ROS can stabilise HIF-1a as mentioned in section 2.3 and can affect the translation machinery to favour HIF-1a production by some unknown mechanism. Surprisingly, this effect of radiation only happens 24 hours after treatment and can sustain for up to one week [55]. This makes using HIF-1 inhibitors in combination with radiotherapy important and necessary. Due to a large number of pharmacological agents available, this review will only focus on discussing one group of molecule Acriflavine that has been discovered recently.

Acriflavine is a multi-heterocyclic molecule that binds to the HIF-1a subunit and inhibits the dimerisation process. The paper published by KangAe Lee et al has proved this to be true since pre-incubating HIF-1b with the drug did not eliminate HIF-1 activity, while pre-incubating HIF-1a did. HIF-1 chromatin co-immunoprecipitation experiment of Acriflavine-treated cells resulted in no PCR product for HRE DNA, while the reverse is true for untreated cells, thus indicating the DNA-binding inhibition hypothesis to be correct. Tumour xenograft showed a reduction in growth after treating with Acriflavine, a positive outcome that can be exploited in future cancer therapy [41].

Due to the upstream position of HIF-1 in many signalling pathways, inhibiting HIF-1 can lead to inhibition of many downstream effectors. This significantly increases the potency of this inhibitor. Nevertheless, the complexity of tumour biology has posed many problems for this approach. According to Moeller, HIF-1 activation by irradiation actually promotes tumour apoptosis, via acting on p53 [53, 54]. Furthermore, some drugs such as Doxorubicin [9] have been shown to increase HIF-1 expression under normoxic conditions, even though it still blocks the binding of HIF-1 to DNA. So will inhibiting HIF-1 lead to a more disastrous cancer or will the beneficial consequences of HIF-1 inhibition outweigh its negative effects? These questions will need to be considered carefully before any decision is made.


4.2. Anti-angiogenic therapy

In contrast to HIF-1 inhibitors, angiogenesis inhibitors have been studied for a much longer period. This could be because the appearance of tumour blood vessels is more readily examined, and also the effect produced by an inhibitor can be more visible. There are many types of angiogenic inhibitors (Figure 8), many of which target the hormone VEGF. Apart from the main activity to trigger blood vessel growth, VEGF is known to trigger many anti-apoptotic pathways including Bcl-2 or Akt/PKB [37]. This protein also inhibits the maturation of tumour-associated dendritic cells [58] and prevents them from presenting tumour antigens to T cells. Endostatin, a tumour angiogenic inhibitor, has been investigated as a potential pharmaceutical agent. A study by Michael S. O’Reilly and colleagues [57] has found the inhibitory effect of recombinant endostatin extracted from Escherichia Coli and baculovirus on both angiogenesis and the development of metastases in Lewis lung carcinoma. However, human trials of this drug so far have not shown much effectiveness with the reason why is still unclear [24].



Figure 8. Different types of angiogenic inhibitor. Many of which are peptide-based molecules and target endothelial cells or VEGF molecule.


One of the most famous achievements in anti-VEGF therapy is the development of monoclonal antibodies, Avastin® or Bevacizumab is one example [24]. This IgG antibody was produced by constantly injecting mice with VEGF to build up an immune response. This antibody was humanised by replacing its constant regions with human components while retaining the antigen-binding sites. The antibody binds and neutralises VEGF, as well as blocks VEGFRs from interacting with their ligands. The antibody was a success and was approved by the FDA in 2004 to treat a wide range of metastatic cancers including non-small cell lung carcinoma and metastatic breast cancer in combination with other methods. The biggest advantage of Avastin is its high specificity with not too severe side effects. Due to its large molecular weight, the antibody can also persist inside the patient’s body for a long period of time. However, because of the large structure, only about 20% [13] of the antibody can make contact with the tumour.

Many of these drugs were tested on immunocompromised (SCID) mice with transplanted human tumours. This sometimes can lead to unexpected results because of the lack of the usual immune-cancer cell interactions found in natural tumours, plus the restriction for cancer to metastasize in these tumour grafts. This could explain why some treatments appear to be effective in mouse models but do not perform very well in human patients. One way to get around this problem is to use multiple different mouse models, combine with more rigorous human trials and statistical analyses. This provides a larger population with less bias, hence limit misleading interpretations.

III. Conclusions

Decades of studying cancer have confirmed the connection between hypoxia, angiogenesis and metastasis. It is the hypoxic microenvironment within tumours that trigger the HIF-1 responses and this together with other mechanisms including JUNB and Ras signalling promotes angiogenesis. The formation of new blood vessels in tumours is an advantage for cancer cells and marks their transformation from being benign to being aggressive. These understandings have shed light on the development of many new anti-cancer agents such as HIF-1 or VEGF-inhibitors. However, there are still many questions that have yet to be answered, such as the significance of oxygen on the activity of cancer cells and how does it affect cancer metabolism in such a way as described as the Warburg effect? What is the mode of action of anti-HIF-1 and anti-angiogenic agents, and how certain treatments can both have positive and negative effects on the state of a tumour? Along with this, hypoxic responses can also be modulated by a non-HIF-1 manner, which leads to a question of how many factors are there, that contribute to the behaviour of tumours. Recently, there are also attempts to indicate the connection between autophagy and the regulation of hypoxia, which can uncover the link between hypoxia and programmed cell death in more details.

The development of in vivo imaging technology will be a major advantage in future research into the microenvironment of tumours. This will allow us to study the system in a more direct way and closest to its natural habitat. The expansion in cancer immunology will also allow us to both optimise previous immunotherapies as well as derive new methods. After all, the how and the why question about cancer metastasis still remain to be fully answered!

References

1 Adams, R. H. and Alitalo, K. (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nature Reviews Molecular Cell Biology. 8, 464-478

2 Basan, M., Hui, S., Okano, H., Zhang, Z., Shen, Y., Williamson, J. R. and Hwa, T. (2015) Overflow metabolism in Escherichia coli results from efficient proteome allocation. Nature. 528, 99-+

3 Balamurugan, K. (2016) HIF-1 at the crossroads of hypoxia, inflammation, and cancer. International journal of cancer. Journal international du cancer. 138, 1058-1066

4 Bergers, G. and Benjamin, L. E. (2003) Tumorigenesis and the angiogenic switch. Nature Reviews Cancer. 3, 401-410

5 Berra, E., Pages, G. and Pouyssegur, J. (2000) MAP kinases and hypoxia in the control of VEGF expression. Cancer and Metastasis Reviews. 19, 139-145

6 Brahimi-Horn, C., Berra, E. and Pouyssegur, J. (2001) Hypoxia: the tumor's gateway to progression along the angiogenic pathway. Trends in Cell Biology. 11, S32-S36

7 Burrell, R. A. and Swanton, C. (2014) Tumour heterogeneity and the evolution of polyclonal drug resistance. Molecular Oncology. 8, 1095-1111

8 Calvo, F. and Sahai, E. (2011) Cell communication networks in cancer invasion. Current Opinion in Cell Biology. 23, 621-629

9 Cao, Y., Eble, J. M., Moon, E., Yuan, H., Weitzel, D. H., Landon, C. D., Nien, C. Y.-C., Hanna, G., Rich, J. N., Provenzale, J. M. and Dewhirst, M. W. (2013) Tumor Cells Upregulate Normoxic HIF-1 alpha in Response to Doxorubicin. Cancer Research. 73, 6230-6242

10 Carmeliet, P. and Jain, R. K. (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature. 473, 298-307

11 Catalano, V., Turdo, A., Di Franco, S., Dieli, F., Todaro, M. and Stassi, G. (2013) Tumor and its microenvironment: A synergistic interplay. Seminars in Cancer Biology. 23, 522-532

12 Chambers, A. F., Groom, A. C. and MacDonald, I. C. (2002) Dissemination and growth of cancer cells in metastatic sites. Nature Reviews Cancer. 2, 563-572

13 Chames, P., Van Regenmortel, M., Weiss, E. and Baty, D. (2009) Therapeutic antibodies: successes, limitations and hopes for the future. British Journal of Pharmacology. 157, 220-233

14 Chandel, N. S., McClintock, D. S., Feliciano, C. E., Wood, T. M., Melendez, J. A., Rodriguez, A. M. and Schumacker, P. T. (2000) Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1 alpha during hypoxia - A mechanism of O-2 sensing. Journal of Biological Chemistry. 275, 25130-25138

15 Christofori, G. and Semb, H. (1999) The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene. Trends in Biochemical Sciences. 24, 73-76

16 Cook, J. and Hagemann, T. (2013) Tumour-associated macrophages and cancer. Current Opinion in Pharmacology. 13, 595-601

17 Cooke, V. G., LeBleu, V. S., Keskin, D., Khan, Z., O'Conne, J. T., Teng, Y., Duncan, M. B., Xie, L., Maeda, G., Vong, S., Sugimoto, H., Rocha, R. M., Damascena, A., Brentani, R. R. and Kalluri, R. (2012) Pericyte Depletion Results in Hypoxia-Associated Epithelial-to-Mesenchymal Transition and Metastasis Mediated by Met Signaling Pathway. Cancer Cell. 21, 66-81

18 DeBerardinis, R. J. (2008) Is cancer a disease of abnormal cellular metabolism? New angles on an old idea. Genetics in Medicine. 10, 267-277

19 Deryugina, E. I. and Quigley, J. P. (2010) Pleiotropic roles of matrix metalloproteinases in tumor angiogenesis: Contrasting, overlapping and compensatory functions. Biochimica Et Biophysica Acta-Molecular Cell Research. 1803, 103-120

20 Doktorova, H., Hrabeta, J., Khalil, M. A. and Eckschlager, T. (2015) Hypoxia-induced chemoresistance in cancer cells: The role of not only HIF-1. Biomedical Papers-Olomouc. 159, 166-177

21 Dvorak, H. F. (2003) Rous-Whipple Award Lecture - How tumors make bad blood vessels and stroma. American Journal of Pathology. 162, 1747-1757

22 Eales, K. L., Hollinshead, K. E. R. and Tennant, D. A. (2016) Hypoxia and metabolic adaptation of cancer cells. Oncogenesis. 5

23 Egeblad, M. and Werb, Z. (2002) New functions for the matrix metalloproteinases in cancer progression. Nature Reviews Cancer. 2, 161-174

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

25 Esteban, M. A., Tran, M. G. B., Harten, S. K., Hill, P., Castellanos, M. C., Chandra, A., Raval, R., O'Brien, T. S. and Maxwell, P. H. (2006) Regulation of E-cadherin expression by VHL and hypoxia-inducible factor. Cancer Research. 66, 3567-3575

26 Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D. and Semenza, G. L. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Molecular and Cellular Biology. 16, 4604-4613

27 Frugtniet, B., Jiang, W. G. and Martin, T. A. (2015) Role of the WASP and WAVE family proteins in breast cancer invasion and metastasis. Breast cancer (Dove Medical Press). 7, 99-109

28 Fulda, S. (2009) Tumor resistance to apoptosis. International Journal of Cancer. 124, 511-515

29 Gerhardt, H., Golding, M., Fruttiger, M., Ruhrberg, C., Lundkvist, A., Abramsson, A., Jeltsch, M., Mitchell, C., Alitalo, K., Shima, D. and Betsholtz, C. (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. Journal of Cell Biology. 161, 1163-1177

30 Gilkes, D. M., Semenza, G. L. and Wirtz, D. (2014) Hypoxia and the extracellular matrix: drivers of tumour metastasis. Nature Reviews Cancer. 14, 430-439

31 Gilkes, D. M., Bajpai, S., Chaturvedi, P., Wirtz, D. and Semenza, G. L. (2013) Hypoxia-inducible Factor 1 (HIF-1) Promotes Extracellular Matrix Remodeling under Hypoxic Conditions by Inducing P4HA1, P4HA2, and PLOD2 Expression in Fibroblasts. Journal of Biological Chemistry. 288, 10819-10829

32 Gupta, G. P. and Massague, J. (2006) Cancer metastasis: Building a framework. Cell. 127, 679-695

33 Harada, H., Kizaka-Kondoh, S., Li, G., Itasaka, S., Shibuya, K., Inoue, M. and Hiraoka, M. (2007) Significance of HIF-1-active cells in angiogenesis and radioresistance. Oncogene. 26, 7508-7516

34 Heinis, M., Simon, M.-T., Ilc, K., Mazure, N. M., Pouyssegur, J., Scharfmann, R. and Duvillie, B. (2010) Oxygen Tension Regulates Pancreatic beta-Cell Differentiation Through Hypoxia-Inducible Factor 1 alpha. Diabetes. 59, 662-669

35 Huang, L. E., Gu, J., Schau, M. and Bunn, H. F. (1998) Regulation of hypoxia-inducible factor 1 alpha is mediated by an O-2-dependent degradation domain via the ubiquitin-proteasome pathway. Proceedings of the National Academy of Sciences of the United States of America. 95, 7987-7992

36 Huang, S. Y., Pettaway, C. A., Uehara, H., Bucana, C. D. and Fidler, I. J. (2001) Blockade of NF-kappa B activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis. Oncogene. 20, 4188-4197

37 Igney, F. H. and Krammer, P. H. (2002) Death and anti-death: Tumour resistance to apoptosis. Nature Reviews Cancer. 2, 277-288

38 Koch, S. and Claesson-Welsh, L. (2012) Signal Transduction by Vascular Endothelial Growth Factor Receptors. Cold Spring Harbor Perspectives in Medicine. 2, 21

39 Koppenol, W. H., Bounds, P. L. and Dang, C. V. (2011) Otto Warburg's contributions to current concepts of cancer metabolism. Nature Reviews Cancer. 11, 325-337

40 Krishnamachary, B., Zagzag, D., Nagasawa, H., Rainey, K., Okuyama, H., Baek, J. H. and Semenza, G. L. (2006) Hypoxia-inducible factor-1-dependent repression of E-cadherin in von Hippel-Lindau tumor suppressor-null renal cell carcinoma mediated by TCF3, ZFHX1A, and ZFHX1B. Cancer Research. 66, 2725-2731

41 Lee, K., Zhang, H., Qian, D. Z., Rey, S., Liu, J. O. and Semenza, G. L. (2009) Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization. Proceedings of the National Academy of Sciences of the United States of America. 106, 17910-17915

42 Liao, D., Corle, C., Seagroves, T. N. and Johnson, R. S. (2007) Hypoxia-inducible factor-1 alpha is a key regulator of metastasis in a transgenic model of cancer initiation and progression. Cancer Research. 67, 563-572

43 Liao, D. and Johnson, R. S. (2007) Hypoxia: A key regulator of angiogenesis in cancer. Cancer and Metastasis Reviews. 26, 281-290

44 Machesky, L. M., Mullins, R. D., Higgs, H. N., Kaiser, D. A., Blanchoin, L., May, R. C., Hall, M. E. and Pollard, T. D. (1999) Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. Proceedings of the National Academy of Sciences of the United States of America. 96, 3739-3744

45 Matsuo, Y., Campbell, P. M., Brekken, R. A., Sung, B., Ouellette, M. M., Fleming, J. B., Aggarwal, B. B., Der, C. J. and Guha, S. (2009) K-Ras Promotes Angiogenesis Mediated by Immortalized Human Pancreatic Epithelial Cells through Mitogen-Activated Protein Kinase Signaling Pathways. Molecular Cancer Research. 7, 799-808

46 Marusyk, A. and Polyak, K. (2010) Tumor heterogeneity: Causes and consequences. Biochimica Et Biophysica Acta-Reviews on Cancer. 1805, 105-117

47 Maxwell, P. H., Dachs, G. U., Gleadle, J. M., Nicholls, L. G., Harris, A. L., Stratford, I. J., Hankinson, O., Pugh, C. W. and Ratcliffe, P. J. (1997) Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proceedings of the National Academy of Sciences of the United States of America. 94, 8104-8109

48 May, R. C., Hall, M. E., Higgs, H. N., Pollard, T. D., Chakraborty, T., Wehland, J., Machesky, L. M. and Sechi, A. S. (1999) The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes. Current Biology. 9, 759-762

49 McDonald, D. M. and Baluk, P. (2002) Significance of blood vessel leakiness in cancer. Cancer Research. 62, 5381-5385

50 Mizukami, Y., Li, J. N., Zhang, X. B., Zimmer, M. A., Iliopoulos, O. and Chung, D. C. (2004) Hypoxia-inducible factor-1-independent regulation of vascular endothelial growth factor by hypoxia in colon cancer. Cancer Research. 64, 1765-1772

51 Mehlen, P. and Puisieux, A. (2006) Metastasis: a question of life or death. Nature Reviews Cancer. 6, 449-458

52 Mizukami, Y., Kohgo, Y. and Chung, D. C. (2007) Hypoxia inducible factor-1-independent pathways in tumor angiogenesis. Clinical Cancer Research. 13, 5670-5674

53 Moeller, B. J., Cao, Y. T., Li, C. Y. and Dewhirst, M. W. (2004) Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: Role of reoxygenation, free radicals, and stress granules. Cancer Cell. 5, 429-441

54 Moeller, B. J., Dreher, M. R., Rabbani, Z. N., Schroeder, T., Cao, Y. T., Li, C. Y. and Dewhirst, M. W. (2005) Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity. Cancer Cell. 8, 99-110

55 Moeller, B. J. and Dewhirst, M. W. (2006) HIF-I and tumour radiosensitivity. British Journal of Cancer. 95, 1-5

56 Olsson, A. K., Dimberg, A., Kreuger, J. and Claesson-Welsh, L. (2006) VEGF receptor signalling - in control of vascular function. Nature Reviews Molecular Cell Biology. 7, 359-371

57 Oreilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R. and Folkman, J. (1997) Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell. 88, 277-285

58 Pollard, J. W. (2004) Tumour-educated macrophages promote tumour progression and metastasis. Nature Reviews Cancer. 4, 71-78

59 Rey, S. and Semenza, G. L. (2010) Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling. Cardiovascular Research. 86, 236-242

60 Reymond, N., d'Agua, B. B. and Ridley, A. J. (2013) Crossing the endothelial barrier during metastasis. Nature Reviews Cancer. 13, 858-870

61 Richard, D. E., Berra, E. and Pouyssegur, J. (1999) Angiogenesis: How a tumor adapts to hypoxia. Biochemical and Biophysical Research Communications. 266, 718-722

62 Rosty, C., Young, J. P., Walsh, M. D., Clendenning, M., Walters, R. J., Pearson, S., Pavluk, E., Nagler, B., Pakenas, D., Jass, J. R., Jenkins, M. A., Win, A. K., Southey, M. C., Parry, S., Hopper, J. L., Giles, G. G., Williamson, E., English, D. R. and Buchanan, D. D. (2013) Colorectal carcinomas with KRAS mutation are associated with distinctive morphological and molecular features. Modern Pathology. 26, 825-834

63 Rundhaug, J. E. (2003) Matrix metalloproteinases, angiogenesis, and cancer - Commentary re: A. C. Lockhart et al., reduction of wound angiogenesis in patients treated with BMS-275291, a broad spectrum matrix metalloproteinase inhibitor. Clin. Cancer Res., 9: 00-00, 2003. Clinical Cancer Research. 9, 551-554

64 Ryan, H. E., Lo, J. and Johnson, R. S. (1998) HIF-1 alpha is required for solid tumor formation and embryonic vascularization. Embo Journal. 17, 3005-3015

65 Schmidt, D., Textor, B., Pein, O. T., Licht, A. H., Andrecht, S., Sator-Schmitt, M., Fusenig, N. E., Angel, P. and Schorpp-Kistner, M. (2007) Critical role for NF-kappa B-induced JunB in VEGF regulation and tumor angiogenesis. Embo Journal. 26, 710-719

66 Semenza, G. L. (2003) Targeting HIF-1 for cancer therapy. Nature Reviews Cancer. 3, 721-732

67 Semenza, G. L. (2004) Intratumoral hypoxia, radiation resistance, and HIF-1. Cancer Cell. 5, 405-406

68 Semenza, G. L. (2012) Hypoxia-inducible factors: mediators of cancer progression and targets for cancer therapy. Trends in Pharmacological Sciences. 33, 207-214

69 Semenza, G. L. and Ruvolo, P. P. (2016) Introduction to tumor microenvironment regulation of cancer cell survival, metastasis, inflammation, and immune surveillance. Biochimica et biophysica acta. 1863, 379-381

70 Semenza, G. L. (2016) The hypoxic tumor microenvironment: A driving force for breast cancer progression. Biochimica et biophysica acta. 1863, 382-391

71 Stoeltzing, O., McCarty, M. F., Wey, J. S., Fan, F., Liu, W. B., Belcheva, A., Bucana, C. D., Semenza, G. L. and Ellis, L. M. (2004) Role of hypoxia-inducible factor 1 alpha in gastric cancer cell growth, angiogenesis, and vessel maturation. Journal of the National Cancer Institute. 96, 946-956

72 Sullivan, R. and Graham, C. H. (2007) Hypoxia-driven selection of the metastatic phenotype. Cancer and Metastasis Reviews. 26, 319-331

73 Suraneni, P., Rubinstein, B., Unruh, J. R., Durnin, M., Hanein, D. and Li, R. (2012) The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. Journal of Cell Biology. 197, 239-251

74 Takenawa, T. and Suetsugu, S. (2007) The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nature Reviews Molecular Cell Biology. 8, 37-48

75 van Uden, P., Kenneth, N. S. and Rocha, S. (2008) Regulation of hypoxia-inducible factor-1 alpha by NF-kappa B. Biochemical Journal. 412, 477-484

76 Wirtz, D., Konstantopoulos, K. and Searson, P. C. (2011) The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nature Reviews Cancer. 11, 512-522

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

78 Yamaguchi, H. and Condeelis, J. (2007) Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochimica Et Biophysica Acta-Molecular Cell Research. 1773, 642-652

79 Yang, M.-H., Wu, M.-Z., Chiou, S.-H., Chen, P.-M., Chang, S.-Y., Liu, C.-J., Teng, S.-C. and Wu, K.-J. (2008) Direct regulation of TWIST by HIF-1 alpha promotes metastasis. Nature Cell Biology. 10, 295-305

80 Yu, B., Shah, A., Wang, B., Rajaram, N., Wang, Q., Ramanujam, N., Palmer, G. M. and Dewhirst, M. W. (2014) Measuring tumor cycling hypoxia and angiogenesis using a side-firing fiber optic probe. Journal of Biophotonics. 7, 552-564

81 Zhang, H., Wong, C. C. L., Wei, H., Gilkes, D. M., Korangath, P., Chaturvedi, P., Schito, L., Chen, J., Krishnamachary, B., Winnard, P. T., Jr., Raman, V., Zhen, L., Mitzner, W. A., Sukumar, S. and Semenza, G. L. (2012) HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs. Oncogene. 31, 1757-1770

82 Zhong, H., De Marzo, A. M., Laughner, E., Lim, M., Hilton, D. A., Zagzag, D., Buechler, P., Isaacs, W. B., Semenza, G. L. and Simons, J. W. (1999) Overexpression of hypoxia-inducible factor 1 alpha in common human cancers and their metastases. Cancer Research. 59, 5830-5835

83 Zhou, J., Schmid, T., Schnitzer, S. and Bruene, B. (2006) Tumor hypoxia and cancer progression. Cancer Letters. 237, 10-21

84 Ziyad, S. and Iruela-Arispe, M. L. (2011) Molecular mechanisms of tumor angiogenesis. Genes & cancer. 2, 1085-1096

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