Can a Drug That Targets an Angiogenesis Factor (VEGF) Be the Next Possible Therapeutic Agent to Stop Growth of Breast

Cancer Tumors?

Stacy Lynn Hernandez

Senior Seminar II September 20, 2007

2 Introduction Cancer is a disease we heard a lot about yesterday and still hear about today. There is still no known cure, but there may be a promising future. Cancer is caused by many different factors. One cause is the stimulation of angiogenesis factors like

vascular endothelial growth factor (VEGF) in tumor cells. Maybe one day we can cure the spread of cancer by using anti-angiogenesis factors in the tumors. In this paper I will discuss a little about cancer and the angiogenesis factor VEGF. Then I will explain a potentially therapeutic agent to help stop breast cancer growth.

What is Angiogenesis? Angiogenesis comes from the Greek words angeion and genesis, which mean vessel and birth (Veile 2005). Judah Flokman first used the term in 1971 (Girolamo 2006). Angiogenesis is the formation of new blood vessels in an already-existing Angiogenesis occurs during embryonic In adults you see angiogenesis during

vascular bed (Wu 2000, Philip 2000). development and is slowed in adults.

menstruation of women in the ovaries and at the site of wounds during the healing process (Girolamo 2006). Angiogenesis is then only seen when triggered by angiogenic mitogens like vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factors. The process of angiogenesis begins with angiogenetic factors being produced and then released to bind to a receptor. This initiates cell proliferation and the migration to begin the growth of new blood vessels. This may lead to tumors being formed if the proliferated cells are damaged or mutated in any way. Angiogenesis goes from normal

3 to pathologic when the blood supply is met but the growth of the vessels will continue (Camp-Sorrell 2003). Below in figure 1 is an example of how the angiogenesis process works.

Figure 1, Angiogenic process. This figure shows how the process of angiogenesis progresses to make new blood vessels (Angiogenesis Foundation 2000).

Background on Cancer One fifth of all deaths that occur in the United States each year are caused by cancer (Lodish). Cancer cells are cells that are genetically damaged in some way, fail to

4 use mechanisms to stop growth (abnormal energy metabolism), and will proliferate autonomously (McKee). These cells fail to use contact inhibition and therefore are malignant and form tumors. The cytoskeleton becomes less organized and reduced. This causes signaling problems. The cells become less adhesive because cell surface molecules are damaged in some way. This is a key factor in metastasis (Lodish). The metabolism of the tumor cells produce glucose from lactic acid in extremely high amounts. This make the tumors pH levels very acidic. The Glycolic rate of tumor cells is high and goes through aerobic glycolysis. This involves oxidative phosphorylation and a form of glycolysis which feeds the tumors along with angiogenesis factors like VEGF (McKee).

Vascular Endothelial Growth Factor Vascular endothelial growth factor is an angiogenesis factor. It has a molecular

weight of about 45 kDa (Carmeliet2005). VEGF is a second protein that is secreted as a dimeric glycoprotein (which is a molecule made by joining two of the exact same molecules, are found on the outer surface in cell membranes and are proteins covalently linked by the nitrogen or oxygen linkages to carbohydrates) by the hemangioblast and the mesenchymal cells near the blood islands (Kliche). Two

disulfide bridges are linked by covalent bonds to a homodimer to form the VEGF molecule. It then binds to receptors hydrophobicly (Kliche). This glycoprotein has the ability to promote vascular permeability, mitogenic activity, and angiogenesis. It is

found in normal tissue such as hepatocytes, visceral epithelium, and the corpus luteum in smaller amounts (Fuckar). Cytokines, hormones and growth factors regulate

5 expression of VEGF in cells (Schrader). Most of secreted VEGF stays in the Once bound to its receptor

extracelluar matrix and on the surface of the cell (Kliche).

on the cell, intracellular signaling is initiated and proliferation and migration begins. There is a family of five VEGF proteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. VEGF-A promotes endothelial cell proliferation, and micro vascular

permeability. VEGF-C and VEGF-D act as lymphangiogenic growth factors. VEGF-E promotes blood vessel dilation and vascular endothelium proliferation. There are a few different mechanisms that have been proposed to explain how VEGF works. One pathway is the focal adhesion kinase (FAK) pathway. In this

pathway VEGF is said to stimulate the increase of cell migration. Another pathway recognized in the mitogenic effects and proliferation of mitogen-activated protein kinase (MAPK) is the protein kinase C pathway. The basement membrane is degraded by enzymes and proteins; this may be partly regulated by nitric oxide (Takimoto2006). Angiogenesis is triggered and cell migration then begins. VEGF may also be regulated by the stimulation of other growth factors, estrogen, and steroids (Ranieri 2006). Knowing all this, once the cells are detected, you now have a direction to look in to finding a cure.

Ways of Detecting Cancer Tumors and Angiogenesis One way to detect cancer is to take a patients blood and look for tumor markers in the blood. One marker is large amounts of VEGF in the blood. You can also measure circulating endothelial cells. There would be an increase in these cells if the patient has cancer. Angiopoietin-1 is another cancer marker (Kiluk).

6 A biopsy can be taken to see if a mast cell is cancerous and to detect biomarkers but it is invasive to the patient. You can detect cancer by an ultrasound and Computed tomography (CT) scans. The other way is by a magnetic resonance imaging (MRI), but angiography is the best way to see larger arteries and veins in the vascular system (Howard-Claudio 2005).

Treatments of Today Surgery is one way to remove the cancer tumors, however, all cells may not be removed and the cancer will reform. Another treatment is radiation treatment. It is given by x-ray machines or a seed of radiation placed in the organ. This treatment doesnt only kill the cancer cells but normal healthy growing cells as well. With most of these treatments it is really a race to kill the cancer before the patient. There is

chemotherapy which is a treatment using an angioplasty agent (Wood). There are not that many effective drugs on the market but many are being explored. All the above treatments are very dangerous to the patients health because they lower the white blood cell count which is part of their immune system. If they get an infection after one of these treatments they may not be able to fight it off and will die. This is why we need a therapy that is less harmless to the patient. Anti-VEGF factors may be the answer.

Possible Therapeutic Agent There are a number of new VEGF antagonist now being developed and tested. One is BAY 43-9006. It directly inhibits the VEGFR-2 and -3. It was shown that in cases of patients with metastatic Renal Cell Cancer (RCC) there was a 25% reduction in 25

7 patients cancer tumor size after a 12-week assessment of 63 patients. There was a stabilization of the cancer in another 18 of the patients. There was some diarrhea, hypertension, hand/foot syndrome and toxic effects that were managed okay. One anti-VEGF oral drug that works on the tyrosine kinase receptor is PYK787. This study had 37 patients of which 17 where stabilized and 6 had partial response. This one caused mild dizziness, vomiting and nausea.

Table1 1. Targeted therapies for VEGF now in development (Rosen 2007).

Agent Bevacizumab Vatalanib (PTK787) Su11248 ZD6747 AG-013736 CP-547632 CEP-7055

Target All VEGF isoforms VEGFR-1, VEGFR-2: PDGFR, C-kit VEGFR-2, PDGFR, C-kit VEGFR-2, VEGFR-3; EFGR/FGFR-1 VEGFR-1, VEGFR-2, VEGFR-3 VEGFR-2; FGFR VEGFR-1, VEGFR-2

Stage of Development Approved Phase III Phase III Phase II Phase II Phase I Phase I

Abbreviations: EGFR=epidermal growth factor receptor; FGFR=Fibroblast growth factor receptor; PDGFR= platelet-derived growth factor receptor; VEGF= vascular endothelial growth factor; VEGFR=vascular endothelial growth factor receptor.

The above table shows the therapies targeted towards VEGF that are currently in development. It shows the name of the drug, the target and the phase of development the drug is in at this time.

Figure 2. Inhibitors and VEGF signaling. This shows the effects of the different protein pathways and at the end of each is the result of the pathway. In red are a few of the anti-VEGF agents that inhibit the pathway to continue in turn stopping the cancer growth (Schrader et al.).

The figure above shows you a little of how Bay 43-9006 and a few other anti-VEGF agents work. This figure shows the protein pathways that VEGF takes to get to the ends of endothelial cell proliferation, vascular permeability, endothelial cell migration and survival. It also shows the VEGF inhibitors: Bay 43-9006, PTK787, Thalidomide, AE-941, Bevacizumab, and others. Each blocks a different pathway and is used

depending on the type of cancer one has and how it grows (Schrader).

9 The figure below shows exactly where the anti-VEGF therapy drugs target on the cell. These are the molecular changes involved (Gille).

Figure 3. This is the potential targets of the antiagiogenesis therapy for VEGF (Gilles).

Bevacizumab There is one drug on the market and in phase III trials now. It is Bevacizumb. It targets all isoforms of VEGF. Bevacizumab is an anti-VEGF recombinant humanized

monoclonal (clone antibody all from same parent) mAb (RhumAb) also known as Avastin or just BEV (Ranieri 2006, Widakowich 2007). BEV neutralizes VEGF isoforms

10 so the endothelial cell proliferation is inhibited. intravenously. The Food and Drug Administration approved it in February 2004 for anti-cancer therapy (Ignoffo 2004). Bevacizumab is administered

Clinical Trials

Preclinical Trials Kim et al. injected nude mice with glioblatoma, human rhabomyosarcoma or cell lines of leiomyosarcoma, which suppressed tumor growth and blood vessel density, but growth rate was not affected. There was another study performed to see if interstitial fluid pressure would be reduced by using and anti-VEGF antibody. This was performed also in nude mice and it was found that the interstitial fluid pressure was reduced and apoptotic cells were slightly increased. The tumors vascular density was reduced significantly because of this antibody (Rugo 2004).

Phase I Trials There have been extensive studies done with Bevacizumab as a monotherapy and also in combination with other drugs or chemotherapy. One early study has shown that Bevacizumab used as a monotherapy showed a 5% to 15% response rate for single agent activity (Mayer 2007).

11 Cobleigh et al reported that in a clinical trial with 75 patients that had breast cancer and were previously treated, were given escalating doses of Bevacizumab as a monotherapy intravenously every other week. They increased the doses from 3mg/kg to 10 mg/kg and finally to 20mg/kg with a maximum of 13 doses all together. The tumors were checked on days 1, 70, and at the end of the trial. The highest response rate was found to be 6.7% of the patients with the 10mg/kg dosage. Out of the 75 patients, 14 required medical attention for hypertension, thrombosis, or mild bleeding (Shih 2006). Bevacizumab was working but not at a high enough response rate to

continue as a monotherapy. The toxicity with this drug alone was very low. Then trials began to combine Bevacizumab with other therapies. A phase Ib trial was conducted

with the addition of chemotherapy and the toxicity related to the chemotherapy was not raised with the addition of the Bevacizumab. This was promising and so phase II trials began with Bevacizumab being used in combination with other therapies (Ignoffo 2004).

Phase II Trials There have been multiple experiments in phase II that have shown an improving response rate. A few phase II trails were conducted at Dana Farber/Harvard Cancer Center. One trial involved 56 patients with metastatic breast cancer and combined the Bevacizumab (10 mg/kg intravenously every 2 weeks) with docetaxel and a response rate of 52% was observed. Another trial performed at this center with 56 patients was given Bevacizumab and vinorelbine (25mg/m weekly). It was observed that the

response rate was 31%. They found in another phase II trial of 55 women who had previously had chemotherapy treatments alone and the response rates were only 10%.

12 The chemotherapy treatments that were combined with Bevacizumab had a response rate of 29% (Mayer 2007). North Central Cancer Treatment Group conducted another study in phase II (NCCTG N0432). They tested 45 patients with Bevacizumab (15mg/kg dose),

docetaxel (75 mg/m) and also added capecitabine (1650 mg/m) in a firstline chemotherapy treatment. After about a median of 7.5 months they collected their

results and 53% of the patients (24) had tumor reduction response. There was a need, however to reduce the drug doses due to a few adverse effects. One patient had Grade 3 hypertension but no patient had any Grade 3 or 4 bleeding (Bando 2007). There was another phase II trial conducted with 75 patients using escalating doses of Bevacizumab in doses of 3mg/kg, 10 mg/kg, and 20mg/kg every other week. Overall, the response rate was 17% at week 22 and 7% more were stable at 1 year (Rugo 2004). There were other phase II trials completed. In all the journals I have read, all the trials have been successful even if the response rate was as low as 6%. This showed me that there is a significant reason why they began phase III trials.

Phase III Trials There was a phase III trial conducted by Eastern Cooperative Oncology Group that used a total of 715 patients with metastatic breast cancer. The trail was conducted using paclitaxal 90mg/m2 with or without Bevacizumab 10mg/kg every two weeks. In this trial progression-free survival (PFS) was measured. The outcome of PFS was

13 greater with the Bevacizumab at 28.2% as opposed without it at 14.2%. The tumor toxicity was not heightened with the addition of Bevacizumab (Gramont 2005). The other large phase III trial was with a chemotherapy called capecitabine. So far this chemotherapy is the only approved therapy for metastatic breast cancer by the Federal Drug Administration to date (Shih 2006). They used 462 women and gave them capecitabine treatments with or without Bevacizumab. They gave capecitabine at doses of 1250 mg/m2 two times a day for two weeks then they had one week rest. The Bevacizumab was given in doses of 15mg/kg every three weeks. The response rate of this trial without Bevacizumab was only 9.1% where as, with Bevacizumab the response rate was 19.8% (Traina 2007, Mayer 2007, Cardones 2006).

Table 2. Phase III data of the drug Bevacizumab with two types of chemotherapy (Traina 2007).

Capecitabine [C] +/Bevacizumab [B] N=462 C C+B RR PFS OS 9.10% 4.17% 14.50% 19.80% 4.86% 15.10%

Paclitaxol [P] +/Bevacizumab [B] N=680 P P+B 13.80% 6.11% 25.20% 29.90% 11.40% 28.40%

Abbreviations: RR, Response Rate: PFS, Progression free survival: OS, Overall survival.

The above table shows the two major Phase III trials conducted with the combination of Bevacizumab with and without either capecitabine or paclitaxol chemotherapy treatments. It shows that the chemotherapy treatments alone do work. When Bevacitzumab is added to either one of the chemotherapy treatments the

14 response rate, progressive free survival and the overall survival rates are significantly higher. This shows me that Bevacitzumab has an enormous impact on the treatment of breast cancer tumors.

Side Effects With the use of Bevacizumab there are four major areas of side effects observed. The severity depended on the dose of the treatment and with which therapy it is combined. The most common side effect has been hypertension. This is due to the production of nitric oxide being reduced when the VEGF is blocked. When a patients nitric oxide is blocked due to the anti-angiogenesis therapy, this lowers the excretion of renal sodium and may cause hypertension also. Patients need to have their blood

pressure monitored regularly when they are taking Bevacizumab. Proteinuria (too much protein in ones urine) is another side effect. Other side effects include arterial and venous thrombosis and bleeding. The percentage of patients with the last three sideeffects were usually very small (Ranieri 2006). It has been observed that Bevacizumab has also impaired the healing of wounds and a patient on this therapy should consider this fact. Also women that are breast feeding should not take Bevacizumab due to the fact that is a category C therapy and is secreted in the breast milk (Shih 2006).

Todays Challenges Even though the anti-VEGF therapy is a very promising therapy and has many positive results, there are always challenges we will face.

15 Today we are looking for a monotherapy. We would like to be able to just use Bevacizumab alone and not in combination with other therapies. Its toxicity is less then other therapies out there and therefore better for the health of most patients. A second challenge is that some tumors may not use angiogenesis but use the existing blood vessels of major organs for blood supply. Today we need better ways of monitoring the anti-angiogenesis factors during treatments. There is a major lack of surrogate markers to track them at this time. This is a future route of study. Another challenge faced is that fact that all tumors are not alike and in one cancerous tumor there may be heterogeneous vascular dependencies. We would need more then one type of therapy for just the one tumor mass. This is another reason biomarkers are a necessity because we need to see how the angiogenesis factors are working. Other challenges that are faced are that some tumors require more blood supply then others and need to be closer to a blood supply. The last major challenge is the fact that all tumor cells have a different level of sensitivity to the VEGF therapy and experiments on different dosing on that patients tumor cells would be required before being administered (Cardones 2006).

Discussion/Conclusion Based on all the trials and research I have done, I have found that the administration of vascular endothelial growth factor does in fact slow and hinder the vascularization of breast cancer tumor cells. The Phase I results of the monotherapy were disappointing because there was not much difference in response rate. The Phase II/III trials and the

16 randomized trials however, were more promising. They have shown that with the addition of chemotherapy the survival rate, response rate and progression free survival rates were dramatically increased. It also shows that that the safety of VEGF therapy is predictable and favorable. With the addition of Bevacizumab with the chemotherapy it has shown that the toxicity of the chemotherapy did not increase. These are all promising facts. There are with every experiment, challenges. If we can work towards overcoming those challenges then the VEGF therapy would be more successful.

Reject or Accept Hypothesis After all the evidence I have written above, I have chosen to accept my hypothesis that a drug targeted against the angiogenesis factor (VEGF) can be the next therapeutic agent in stopping the growth of breast cancer tumors.

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