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Cancer is a disease that is able to affect every person in some form during his or her lifetime. Unfortunately, some of the most common cancers still have very high mortality rates, as the main treatments are not very effective. Lung cancer for example accounts for 22% of all cancer-related deaths and 7% of all deaths in the UK, which totals at 30,301 people (Office for National Statistics, 2008). The need for more advanced, more specific and faster acting anti-cancer therapies is crucial to pharmaceutical companies around the world.
Surgeons and doctors around the world have conducted much of the research into this area, as they have the first contact with cancerous cells and can recognise patterns and anomalies. In 1971, Dr. Judah Folkman noticed that tumour growth was angiogenesis-dependent, and that solid tumours became much more aggressive and metastatic following an increased blood supply (Folkman, 1971). His findings were very exciting within the field of cancer therapy because over 90% of all cancers present as solid tumours (Abdelrahim et al., 2010). Folkman was one of the main pioneers involved with angiogenesis targeting anti-cancer therapy, and along with his most recognised discovery in 1971; he also discovered the first anti-angiogenic compound in cartilage (Brem & Folkman, 1975).
In this article I would like to discuss the natural mechanism of angiogenesis, involving a number of the intricate pathways dominating the process, highlight some of the key molecules recognised as mediators in natural angiogenesis and recognise how tumour vasculature differs in its structure and molecular pathways. With a solid understanding of the mechanisms involved, my aim is to introduce a few of the most effective treatments available and their mechanisms, as well as looking to the future development within this area of cancer therapy.
Natural angiogenesis is a highly regulated process and is key to post-natal development, wound healing, menstruation and ovulation within the human body (Dhanabal et al., 2008; Papetti & Herman, 2002). All human cells are required to be within 100 to 200Âµm of blood vessels in order to receive adequate nutrients and oxygen to survive and if cells receive less than their adequate supply of oxygen it is known as hypoxia, a lack of nutrients is known hypoglycaemia (Carmeliet & Jain, 2000; Benjamin & Keshet, 1997). Hypoxia-inducible factors such as HIF-1ï¡, HIF-1ï¢ and HIF-2ï¡ are released during this phenomenon and trigger a large molecular cascade to initiate angiogenesis in order to relieve oxygen tension through increased blood supply to the area (Carmeliet, 2000; Kappers et al., 2009).
The supply of blood to cells within the body is reliant on the proliferation and migration of endothelial cells, the cells that line blood vessel walls, vital in vascular tissue (Alberts et al., 1994; Karamysheva, 2008). Development of vascular tissue is dependent on the balance between pro-angiogenic and anti-angiogenic signals (Karamysheva, 2008; Carmeliet & Jain, 2000). The increased transcription of pro-angiogenic factors including VEGF, its receptors, angiopoietins, transforming growth factors (TGFs), fibroblast growth factors (FGFs) and many other molecules, over anti-angiogenic mediators, sways the balance in favour of vascular expansion via increased expression of vascular endothelial growth factor (VEGF), this is called the 'angiogenic switch' (Carmeliet, 2000; Karamysheva 2008; Semenza, 2001).
Figure 1. Stages in natural angiogenesis. (1) Ang2 destabilises the vessel. (2) VEGF release increasing vessel permeability. (3) VEGF, EGF and FGF promote endothelial cell proliferation. (4), migration of endothelial cells by VEGF and FGF. (5) Cells attach and strengthen together. (6) Hollow out the tube to form vessel-like structure, via TNF-ï¡-induced cell apoptosis. (7) Improving structure and strength (8) of the new vessel. (9) Stabilisation of the final structure. Image courtesy of Papetti and Herman (2002). TGF-ï¢, transforming growth factor-ï¢.The destabilisation of the existing capillary walls in the capillary plexus, via removal of pericytes, is involved in the initial stages of angiogenesis. Pericytes are fundamental in vasculature tissue as they stabilise and strengthen endothelial cell connections. VEGF binds to VEGR2 on the cell surface of endothelial cells and increases the permeability of the vasculature through matrix metalloproteinase (MMP) activation (Klagsbrun & Moses, 1999). The involvement of angiopoietins (Ang-1 and Ang-2) with the receptor Tie2 is also known to aid this process. However, they have different effects on the Tie2 receptor because Ang-1 shows agonistic effects, whereas Ang-2 is antagonistic. It has been found that Ang-1 in natural angiogenesis induces chemotaxis of endothelial cells and is involved in the development of pericytes from mesenchymal cells, as well as having large involvement in the latter stages: decreasing vascular permeability, thus stabilising the maturation of the vascular system (Karamysheva, 2008; Papetti & Herman, 2002). In contrast, Ang-2 promotes vascular destabilisation and is important in vascular remodelling (Makrilia et al., 2009).
To prevent destabilisation along the whole of the original vessel 'Notch signalling' enables some endothelial cells to act as 'stalks' and others as specialised 'tip' cells, which are guided by VEGF concentrations, towards where it is required (Zen & Madeddu, 2009). The 'Notch signalling' pathway is a very complex pathway involving a vast number of mediators.
In the latter stages of development, endothelial cells in their proliferative phase hollow out and form tubes for blood to pass through, with the aid of tumour necrosis factor (TNF-ï¡). The stabilisation of the structure and decrease in proliferation allows cell-to-cell bonds to strengthen (Papetti & Herman, 2002).
Angiogenesis has a crucial in the development of many human diseases such as rheumatic disease, psoriasis and cancer (Cao, 2001; Dhanabal et al., 2008). Among these diseases and in natural angiogenesis VEGF has been identified as the key mediator in triggering neo-vascularisation.
The importance of VEGF:
Figure 2. Image, courtesy of www.researchvegf.com. Shows VEGF binding to VEGFR2, leading to multiple tyrosine kinase cell signalling pathways: phosphoinositol-3-kinase pathway (PI3K) to promote vascular permeability and endothelial cell survival, p38MAPK for endothelial cell migration and Raf/Mek/Erk pathway for mediating endothelial cell proliferation.VEGF and its isoforms have been shown to act directly on vascular endothelial cells. Its other functions include the stimulation of their endothelial cell growth and increasing expression of Bcl-2 and A1, anti-apoptopic proteins (Gerber et al., 1998; Ferrara, 2004). It does this by binding to VEGF receptors VEGFR1, VEGFR2 and VEGFR3, which all have differing roles: VEGFR1 primarily in embryonic growth, VEGFR2 in the greatest proportion of VEGF-related events and VEGFR3 in lymphangiogenesis (Hicklin & Ellis, 2005; Ferrara, 2004).
The actions of VEGFR2 have been recognised as the most crucial in the processes of angiogenesis, particularly in relation to tumour angiogenesis and its possible therapeutic targets. As seen in Figure 1 VEGFR2 has been found accountable for the phosphorylation of three signalling pathways: the PI3K signalling pathway which increases the vascular permeability and enhances the chances of the cell's survival, the Raf/Mek/Erk pathway accountable for endothelial cell proliferation and the p38MAPK pathway which enables chemotaxis of endothelial cells (Gerber et al., 1998; Ferrara, 2004).
It is now well known that solid tumours require angiogenesis to survive and grow. A solid tumour cannot grow over approximately 3mm3 without the creation of new blood vessels, which prevents tumour malignancy and renders the mass harmless to the person (Ferrara, 2004; Gimbrone et al., 1972; Abdelrahim et al., 2010). VEGF expression has been singled out as the rate-limiting step in tumour angiogenesis (Ferrara, 2004; Dhanabal et al., 2008; Carmeliet, 2000). Development of vascular tissue to provide tumour cells with oxygen and nutrients allows them to grow and metastasise, thus effectively making VEGF itself, an anti-apoptopic factor.
Figure 3. Shows the epigenetic and genetic factors affecting release of VEGF and its isoforms. Also visible is the receptor that they bind to, and the cell signalling pathways that interaction with VEGFR2 would mediate. Image, courtesy of Kerbel (2008).Many tumour cell lines have been found to increase expression of mRNA for VEGF, such as in the lung, breast tissue, kidney and gastro-intestinal tract (Ferrara, 2004). This acts in a paracrine manner, where the tumour cells have the ability to release the chemical but do not respond to it directly as they often do not have the receptors (Kerbel, 2008). However, Fukumura et al., (1998) found, using green fluorescent protein for VEGF in transgenic mice that stromal cells, connective tissue associated with the tumour, also release VEGF. This suggests that gene mutations in tumour and surrounding tissue lead to increased transcription of VEGF mRNA. Surrounding and supportive tissues play a very large role in tumour angiogenesis and their complex signalling is key to tumour growth. These molecules largely consist of endothelial cells, tumour-associated macrophages, pericytes, fibroblasts and stromal cells (Faivre et al., 2007).
Oncogenes play an important role in the up-regulation of VEGF from tumour cells. In von Hippel-Lindau (VHL) Syndrome a mutation of the VHL tumour suppressor gene leads to an increase in vascularisation in patients and an increased risk in developing certain types of cancers. The VHL gene is also vital in tumour angiogenesis and has been shown to induce hypoxia-inducible factors HIF-1ï¡ and HIF-2ï¡ (Harris, 2000; Kerbel, 2008). This is because its normal action on HIF-1ï¡ and HIF-2ï¡ is to contain their expression (Rini, 2007). As described earlier in this article these two substrates are involved in the regulation of natural angiogenesis, through altering the balance between pro- and anti-angiogenic factors, increasing VEGF transcription. Other oncogenes that show correlation between their expression and the expression of VEGF include p53 (Montero et al., 2007) and mutated PTEN-suppressor genes (Ma et al., 2009), among others.
Figure 4. From Jain (2001). (a) A representation of what normal angiogenesis vessel structure should be. (b) Shows excessive angiogenesis, seen in tumour growth. (c) Demonstration of how (b) could become more effective in administering cytotoxic drugs. (d) Depicts the inadequate blood flow to tumour vasculature, which is commonly seen.In many cases the structure of tumour vasculature has been described as very messy, because of the effects of VEGF over-expression. An increase in VEGF expression increases the vascular permeability of capillaries via VEGF binding to VEGFR2 on endothelial cells (Ferrara, 2004). When VEGF expression is unregulated vascular permeability can take place in many different, and unnecessary locations, resulting in hyper-permeability. The other effects of VEGF allow the formation of new vascular tissue, however, their structure is haphazard and poorly organised through over-production of endothelial cells. This affects the balance between interstitial and vascular blood pressure. It also determines the tumour's ability to receive blood and can have very contrary effects through forming inadequate blood vessels with a very inadequate blood supply. This, in turn, can have serious effects to the delivery of cytotoxic cancer therapies (Jain, R.K, 2001).
Drugs targeting tumour angiogenesis:
Bevacizumab is currently used in the treatment of renal cell carcinoma (RCC), which is normally found in the proximal convoluted tubule of a nephron. This particular type of cancer has a high incidence rate and is among the top three genito-urinary cancers, following prostate and bladder cancer (â€¦..) . Also in non-small cell lung cancer (NSCLC), breast, glioblastoma and metastatic colorectal cancer. It is the first-line choice of treatment for both non-small cell lung cancer and colorectal cancer, which are both in phase IV of clinical trials, having been approved by the FDA. It also has an approved clinical trial phase IV in the treatment of HER2 negative breast cancer. This trial has been approved but is not yet active, and would include co-administration with docetaxel, an anti-mitotic agent (U.S National Cancer Institute). Research into the effectiveness of the drug in vivo suggests that it is much more effective when co-administered with other drugs involved in chemotherapy (Ferrara et al., 2004), however, some trials have found this not to be the case. In a randomised trial, Bevacizumab was paired with cetuximab to counteract colorectal cancer but found that it decreased patients overall probability of survival (Tol et al., 2009).
The half-life of Bevacizumab varies between 17 and 21 days and there has not been any citing in the emergence of antibodies against Bevacizumab in the human body, which is extremely useful during treatment (Ferrara et al., 2004). This drug is an example of a humanised recombinant monoclonal antibody produced by Genentech, which means that complementary determining regions of mouse DNA are converted to human DNA, making the likelihood of an immune response to lower (Ferrara et al., 2004; Presta et al., 1997).
Figure 5. The molecular action of Bevacizumab, Sorafenib and Sunitinib. Illustration of the binding of Bevacizumab to VEGF, to prevent VEGFR2 stimulation. The action of tyrosine kinase inhibitors, Sorafenib and Sunitinib, where Sorafenib is shown to inhibit both the Raf/Mek/Erk and PI3K pathways. Sunitinib inhibits the PI3K pathway. Image, courtesy of Rini (2007).The mechanism of Bevacizumab is to bind and deactivate VEGF. It has been found to bind to all of VEGF's isoforms as well as its proteolytic fragments via the Gly88 binding protein on its Fab sequence (Ferrara et al., 2004). Bevacizumab is effective through inhibiting the initiation of tumour angiogenesis, which keeps the tumour cells in a hypoxic and hypoglycaemic state and eventually triggers apoptosis through cell malnourishment. This denotes both the cytotoxic and cytostatic potential of Bevacizumab (Rini, 2007), and is the reason why it is used in conjunction with other cytotoxic agents, specifically 5-fluorouacil (5-FU) in colorectal cancer (Gerber & Ferrara, 2005). It also signifies that the drug does not necessarily act on tumour cells, but rather on surrounding endothelial cells, preventing vascularisation of the tumour (Kim et al., 1993).
As with many cancer treatments this anti-VEGF therapy was found to have a few negative side effects. However, a few are of serious risk, even if they are extremely rare, and some deaths were seen during clinical trials. According to www.Avastin.com, provided by Genentech, [accessed on 22/03/10] serious side effects included severe bleeding, which was characterised by nosebleeds, coughing up blood and bleeding in stomach. Other severe problems included gastrointestinal perforation and wound healing problems. More common side effects were skin rashes, high blood pressure and proteinuria (Ferrara et al., 2004). Across all trials with Bevacizumab, up to 21% of patients were permanently removed from treatment due to side effects (www.avastin.com [accessed 22/03/10]).
Nexavar originally marketed Sorafenib under the name BAY 43-9006. The drug is given orally and is currently in phase IV clinical trials for renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), which is one of the deadliest types of cancer, and gastrointestinal cancer. It is also in phase III trials for adenomas of the pancreas and NSCLC (US National Cancer Institute) among other trials involving solid tumours of earlier phases (Wilhelm et al., 2008).
Sorafenib has a very complex mechanism of action and has found to be an inhibitor at multiple tyrosine kinase sites. It is a small molecule inhibitor, which means that the organic compound that is being targeted has a low molecular weight and is typically found in cell signalling pathways. Initially it was thought to only inhibit Raf serine/threonine kinase isoforms within tumour and endothelial cells, however, with further investigation it was found to have an inhibitory effect on a variety of Raf isoforms. These include Raf-1, wild-type B-Raf and oncogenic B-Raf V600E, of which the potency of its effect decreases respectively (Wilhelm et al., 2008). It was also originally thought to have no effect on Mek-1 or Erk-1, two signalling molecules further in the cascade in vitro, but was shown to decrease their activity in vivo (Wilhelm et al., 2004; Wilhelm et al., 2008). Furthermore, Sorafenib has illustrated inhibitory effects on PDGFR-ï¢, VEGFR1, VEGFR2 and VEGFR3 in vitro (Wilhelm et al., 2008). This may be the cause of Sorafenib-induced apoptosis, however, it is believed that there is another reason for this, due to the decreased transcription of anti-apoptopic protein myeloid cell leukaemia-1. Unfortunately, this is not been fully established, but is a common theme in tumour cell apoptosis (Wilhelm et al., 2008).
The negative effects associated with Sorafenib, which is taken orally, twice a day include minor skin rashes, fatigue, high blood pressure, alopecia and diarrhoea. Unfortunately, more serious side effects do occur, which are deemed medical emergencies. These include myocardial infarction and severe bleeding; in these cases treatment with Sorafenib would be discontinued (www.Nexavar.co.uk [accessed 23/3/10]).
Sunitinib malate is a drug produced by Pfizer. It also known as SU11248, and is marketed under the name Sutent. Sunitinib is FDA approved to treat gastrointestinal stromal tumours (GIST), after a significant improvement in time to progression compared to placebos in a double-blind test (Blay, 2010). It is also approved to treat metastatic renal cell carcinomas (RCC), following an improvement in progression-free survival from observation of Sunitinib against treatment with interferon-ï¡ (IFN-ï¡) (US National Cancer Institute [accessed 23/3/10]).
The mechanism of Sunitinib acts against both angiogenesis and tumour activities. It is a small molecule multi-targeted tyrosine kinase inhibitor, affecting VEGFR1, VEGFR2, VEGFR3, PDGFRï¡, PDGFRï¢ and stem cell growth factor receptor (KIT) (Blay, 2010; Faivre et al., 2007; Abdelrahim et al., 2010). The effects this has on tumour cells is to indirectly suppress their activity through preventing endothelial cell mediated migration and proliferation, as well as other molecular signals that trigger angiogenesis. However, it also has direct effects on tumour cell growth, proliferation and survival through inhibition of the receptor tyrosine kinase pathways (Abdelrahim et al., 2010). The proposed mechanism for direct Sunitinib-induced tumour necrosis is through morphological changes to endothelial cells, separating them from each other, causing congestion within the blood vessel. Platelets and red blood cells coagulate leading to marked decrease in blood flow, starving tumour cells of oxygen and nutrients, ultimately leading to their death (Faivre et al., 2007).
The safety concerns seen with Sunitinib are similar to that of Sorafenib, and the majority of ill effects seen were of mild severity. More severe negative effects of taking the drug during clinical trials included neutropaenia, thrombocytopaenia and anaemia. However, discontinuation because of the side effects was very uncommon as only 9% of phase III GIST patients given Sunitinib discontinued drug use, compared to 8% given placebos (Faivre, et al., 2007).
Conclusions and future directions:
Anti-angiogenic drugs were hailed to be the next great step towards cancer treatment, but how successful have the new therapies been?
Much of the progress of anti-angiogenic targeted therapies has been overshadowed by their ineffectiveness to cure the problems caused by tumours and has thus led to many of them being administered in conjunction with other cytotoxic agents, for example Bevacizumab with docetaxel. Instead, much of the improvements witnessed have not been because of their ability to cure the disease, but only to prolong it (Loges et al., 2009). It has been suggested that improving the structure of tumour vasculature may be another way of improving treatment in conjunction with cytotoxic agents. Therefore, the cytotoxic agents have more reliable contact with tumour cells and have the potential to induce more tumour cell death (Jain, 2001).
Misunderstanding the method of each drug's action is a reason for the increased use of biomarkers, both for diagnostic means and to predict the pharmacodynamics of drugs (Murukesh et al., 2010). One of the proposed mechanisms of resistance to treatment with Sunitinib involves mutations in tyrosine kinase receptors, which would lead to ineffective action from other tyrosine kinase inhibitors that are currently in use, such as imatinib (Faivre et al., 2007).
Another suggested mechanism is that the tumour increases its release of VEGF to compensate for the lack of angiogenesis occurring through tyrosine kinase receptor block and VEGF inhibition. This could have more marked effect on other surrounding cells involved in mediating tumour angiogenesis. A natural reaction for oxygen and nutrient starved cells would be to increase expression of HIFs. Therefore when an anti-angiogenic agent is given to inhibit this, the immediate response would surely be to increase expression of HIF-1ï¡ and HIF-2ï¡ to try and stimulate a more aggressive 'angiogenic switch' (Loges et al., 2009; Brahimi-Horn et al., 2009). It has been shown that tumour cells are better equipped when dealing with hypoxia than their surrounding cells, and that hypoxia effectively acts to select more metastatic cells, that are more likely to evade anti-angiogenic therapies (Brahimi-Horn et al., 2009; Yu et al., 2002).
With a greater understanding of the role and action of each molecule in each pathway, the more relevant and effective anti-angiogenic therapies have the potential to be. I feel a flaw in the development of anti-angiogenic drugs was the extremely high expectations on the new therapy. Perhaps more subtle and indirect approaches would have had greater benefit for patients. Therefore, using angiogenesis inhibition in order to make surgery or radiotherapy more effective by isolating the tumour from unaffected cells. I believe there is still a vast amount of research to be done in tumour angiogenesis and the considerable amount of work that has already been done will benefit cancer patients greatly in the years to come.