Breast Cancer and Biotechnology Jacquie Bay, Jo Perry, Michal Denny and Peter Lobie

LENScience Senior Biology Seminar Series Breast Cancer and Biotechnology Jacquie Bay, Jo Perry, Michal Denny and Peter Lobie Breast Cancer Each year in New Zealand, approximately 2,400 women and 20 men are diagnosed with breast cancer and approximately 600 people die of breast cancer 1. Over the last 30 years, the number of people diagnosed with breast cancer increased, however very recently we have been seeing a decline in incidence in many countries which is most likely due to identification of Hormone Replacement Treatment as a risk factor. In 1960, around 1 in 30 NZ women were diagnosed with breast cancer in their life time; today that figure is 1 in 9. This is partly due to improvements in screening and diagnosis but breast cancer rates were also increasing. A woman in NZ has an 11% chance of being affected by breast cancer. While the number of women being diagnosed with breast cancer has been increasing, the good news is that the death rate from breast cancer decreased by 27% in the past decade 1. This is a result of increased understanding of the disease, improved screening, diagnostic methods, and treatment. New Zealand has a large and active cancer research community that plays an important and often world leading role in cancer research. Biotechnology has played a significant role in the development of knowledge of cancer and treatment of this disease. What is cancer? Healthy cells have a fixed life span. They replicate (reproduce) a certain number of times and then enter a period of senescence where they stop dividing. This is followed later by cell death (apoptosis). Cancer occurs when cells keep growing instead of dying. This causes the development of tumours which disrupt normal body function. Cancer may occur in any part of the body. Tumours may be solid masses of cells such as a breast cancer tumour, or they may be liquid tumours consisting of blood cells that have become cancerous such as in lymphoma and leukaemia. The cell cycle (Fig 1) controls normal growth and reproduction in cells. It is controlled by two families of molecules. Faults during the cell cycle such as inadequate growth and mistakes in the replication of the chromosomes are generally picked up by these control agents. The cell will then be repaired, or stop dividing and die later or the cell will self destruct (apoptosis). The process of cell growth and division is regulated by signals from inside and outside the cell. Gap 2 The cell will grow again and the proteins that are required during mitosis will be made. At the end of this phase there is another check point to ensure that everything is correct before cell division. Mitosis The cell divides to produce two identical daughter cells. The Cell Cycle. Controlling cell growth and reproduction Synthesis The DNA is copied in readiness for cell replication. Identical copies of all chromosomes are synthesised. Gap 1 The cell grows, produces RNA, & synthesises proteins. A check system ensures that the cell is ready for DNA synthesis. If the check finds that there is a problem, the cell will be destroyed. Fig 1. The Cell Cycle 1

Unlike normal cells, cancer cells are immortal. They do not respond correctly to the regulatory signals and will go on replicating themselves over and over again. This fast rate of reproduction creates a mass of excess cells, called a tumour. What makes a cell become cancerous? Cancers arise from changes in the cell that give the cell a growth advantage. This means the cell has an increased ability to grow faster and more aggressively. The changes may be in the DNA sequence (mutations) or they may be epigenetic changes such as DNA methylation, that alter gene expression without altering the sequence of base pairs in the gene. DNA methylation can be caused by changes in hormone exposure, or exposure to chemicals in the environment and in diet. (You learnt about epigenetic changes in the first seminar Understanding Gene Expression ). Cancers that start with a mutation are usually a result of exposure to an environmental factor (a mutagen) but they can also be a result of errors in everyday cellular process e.g. DNA replication errors. They can also occur spontaneously. In most cases, the mutations are caught in the checks that are a part of the cell cycle and either the error is corrected or the cell is destroyed. If the cell cycle is not carefully controlled and checked, the mutated cell will survive and produce more mutated cells. Cancer is usually the result of an accumulation of mutations or changes in gene expression that transform the normal cell into a cancerous cell. As we get older, we accumulate more mutations and changes in the DNA packaging, therefore increasing the chances of cancer occurring as we age. The Hallmarks of Cancer Scientists Douglas Hanahan and Robert Weinberg 2 think that all the different types of cancers share six essential changes in the physiology of the cell. These changes all contribute to the production of a successful tumour. They called these six essential characteristics the Hallmarks of Cancer (Fig. 2). 1. Uncontrolled Growth Normal cells start or stop growing in response to signals. Cancer cells do not respond to these signals. They will grow when there are no growth signals and continue to grow when they are getting stop signals. So no matter what colour the traffic lights are, cancer cells will go! 2. Evading Death Normal cells are meant to die when they get old or damaged. Signals in the cell start a process where the cell membrane is disrupted and enzymes break down the cell contents, which are then engulfed by neighbouring cells. This is called programmed cell death or apoptosis Cancer cells avoid programmed cell death. They have the ability to repress (ignore) the signals that start the process of apoptosis. Evading Death Angiogenesis Fig 2. The Hallmarks of Cancer (After Hanahan & Weinberg, 2000) Uncontrolled Growth Becoming Immortal Promoting Mutations Invading Tissues & Avoiding Detection 2

This means that all cells must be in close proximity (within 100µm) of a capillary if they are to survive. Growth of a tumour is not possible past a certain size unless the cells are capable of attracting blood vessels to grow into the tumour in order to supply nutrients and remove wastes. We say a tumour is Angiogenic if it has the ability to attract blood vessels to grow into it. 4. Becoming Immortal Normal cells have a limited number of times that they can divide before they stop growth. Sequences of DNA on the ends of chromosomes called telomeres are responsible for making sure that chromosomes do not fuse end to end during cell division. Each time a cell divides, the telomeric DNA get slightly shorter. When it gets so short that it cannot protect the chromosome anymore the cell dies. In cancer cells the telomeric DNA does not get shorter because an enzyme called telomerase is released that lengthens the telomeres so the cell can keep on replicating endlessly. 5. Invading other tissues and avoiding detection (Metastasis) It is common for cancers to move from the site of the primary tumour to other parts of the body. This is called tissue invasion or metastasis. The immune system is constantly on the look out for cells that are foreign or do not belong. Cancer cells often look different to normal cells and when spreading out will invade tissues different to their own. In order to survive, pre cancer cells and cancer cells must evade detection by the immune system. 6. Promoting Mutations The development of cancer requires an accumulation of mutations in a number of genes over a period of time. Some of these changes accelerate the rate at which mutations occur meaning that mutations are acquired at a faster rate. Together all these characteristics contribute to the progression of normal cells into cancer (Fig 3.) Normal Cell Uncontrolled Growth Capillary Invasion Angiogenesis Cancer Cells Poor Prognosis Metastasis Invasive Cancer Cell Fig 3. Cancer Progression Risk Factors for Cancer Growing older Tobacco Sunlight Ionizing radiation Certain chemicals and other substances Some viruses and bacteria Certain hormones Family history of cancer Alcohol Poor diet, lack of physical activity, or being overweight 3

Breast Cancer and Human Growth Hormone Human growth hormone (hgh) is made and secreted by the pituitary gland, which is located at the base of the brain. Human growth hormone is essential for normal growth and development in humans. Not enough hgh in childhood results in a person who is very short while excess growth hormone results in gigantism. In addition to the endocrine hgh that is produced and secreted from the pituitary gland, hgh is also produced and secreted locally in different sites around the body. (Locally means the hormone affects only that area). These sites include parts of the central nervous system, cells within the immune system, blood vessels and breast tissue. The levels of hgh secreted from the pituitary gland fluctuates during the day. It has high peaks and low troughs. In contrast, the hgh that is secreted from the local tissue sites such as breast tissue, is secreted continuously at very low levels (Fig 4). Studies conducted at Liggins Institute at the University of Auckland have demonstrated that hgh secreted locally in the breast tissue plays a critical role in the development of breast cancer 3. Autocrine hgh secreted in the breast is essential for normal breast development during puberty. However the Liggins scientists have found that there are increased levels of autocrine hgh found in breast cancer tumours. Cell Culture You may be surprised to learn the human body cells are able to grow outside our bodies if they are given the right conditions (e.g. specific ph, temperature, and growth factors) and the nutrients they need to stay alive. It s not quite as easy as it sounds as the right conditions require a controlled laboratory environment, especially to prevent infection by bacteria or fungi. Cells grown outside of the whole organism are said to be growing in vitro. This literally means in glass. (Cells inside an organism are said to be in vivo or in life ). 4 Definitions Endocrine: hormones that are secreted into the bloodstream and carried to the target tissues or cells Autocrine: hormones that only act the type of cell that produced it Fig 4. Excretion of Growth Hormone in Humans Human cells, including cancer cells, can be cultured outside the human body in plastic flasks kept in incubators. Cells grown this way are called a cell culture. Cell cultures allow scientists to test the effect of chemicals on the cells and to investigate the normal physiology or biochemistry of cells. There are many genes that scientists have noted are expressed differently in cancer cells. Once a gene has been identified it can be introduced to a cell line and the subsequent effects on the cells studied by growing these cells in cell culture. The gene must first be cloned and inserted into a plasmid vector and then amplified in bacteria. The vector is then transferred into mammalian cells using cell transfection technologies such as liposomes. These cells are incubated at human body temperatures and supplied with nutrients. By comparing the gene expression in these cells with control cells scientists can determine the changes in the cells that have been caused by the inserted gene. An alternative approach to look at the effects of a single gene is to inhibit the function of the gene or protein if the cells already express it.

The Liggins scientists wanted to know if these increased levels of autocrine hgh were linked in some way to the development of breast cancer. To find this out they used cultured human breast cells to study the effect of differing levels of hgh on the development of breast cancer. The scientists found that when hgh is being secreted from breast cancer cells (i.e. autocrine hgh) it increases cell growth rates and the cells are more invasive (Fig 5 and 6). Fig 6: Autocrine hgh production in cells increases number of cancer cells. Control: no hgh Cells producing hgh Fig 5: The control cells on the left have no growth hormone being secreted. The cells on the right have the hgh gene inserted and are secreting autocrine hgh. They are growing more aggressively. Next the scientists wanted to find out about the molecules that are regulated by hgh that were causing the cells to grow faster and more aggressively. Their suspicion was that the autocrine hgh was affecting the expression of genes in the breast cancer cells. Gene expression what is going on to create this phenotype? When studying disease, scientists want to find out how gene expression is being affected in the cells (e.g. genes being switched on/off or increasing or decreasing the level of gene expression). The phenotypic response that we see in organisms, such as the development of breast cancer, is almost always the result of a group of genes being expressed together, rather than just one gene. Scientists have been able to study gene expression one gene at a time for many years using a biotechnological process called the Polymerase Chain Reaction or PCR for short. PCR is used to amplify (make multiple copies of) DNA. In gene expression studies PCR is used to amplify specific genes to find out whether that gene is being expressed in the tissue being studied. If genes are being expressed then mrna will be produced. This RNA is extracted from the cells and a process called reverse transcriptase is used to make a short section of DNA that is complementary to the mrna called cdna. The cdna is slightly different from the original DNA because it does not contain introns. The amount of cdna produced will depend on the amount of mrna which is determined by how active the gene is. 5 Using PCR Technologies RNA is extracted from the cells *Reverse Transcriptase reaction produces cdna from the RNA The Polymerase Chain Reaction (PCR) uses specific primers to produce multiple copies of the target cdna The target DNA is analysed using Gel Electrophoresis. A variation on PCR called REAL TIME PCR uses fluorescent labels and provides a quantitative analysis of the PCR product. *Reverse transcriptase is literally the reverse of transcription in protein synthesis it takes mrna and creates DNA.

The cdna is then amplified and analysed to establish how much mrna was in the original sample. This gives an indication of how active the gene was. PCR only allows the study of one gene at a time, which makes the process very slow. The development of a new biotechnology, microarrays had a major impact on research into gene expression because they allow scientists to study thousands of genes all at the same time giving the ability to study a genetic profile. Using a microarray we can identify which specific genes a cell is using at a particular point in time. This means that we can compare which genes are turned on or off in different conditions (e.g. when cancer is present compared to when cancer is absent). What is a Microarray? A microarray is a small glass slide that contains tiny fragments of known DNA sequences in different spots on a slide. This is also known as a gene or DNA chip. A Human Genome microarray will contain small fragments of each of the genes in the genome. These are called probes. Each spot on the slide contains multiple copies of the same probe. The results of a microarray The Experimental Model First cdna is extracted from the cultured cells and fluorescently labelled. When the labeled cdna solution is washed over the slide, the fluorescently labeled cdna pieces that match the complimentary base pairs on the slide will bond. When the slide is washed, the bonded cdna fragments will remain in place and the other fragments will wash away. The fluorescent spots on the slide are read are read using a microarray scanner and the levels of fluorescence intensity analysed with specialized software. The scientists suspected that the hgh produced and secreted by the breast cells was changing the expression of genes in the cells that controlled the cell cycle. To test this the scientists created two cell lines. One contained the gene for hgh the other (the control) produced no hgh (Fig 7.). To get the gene into the cells the scientists used biotechnological techniques. First the hgh gene was isolated and multiple copies made using PCR. The hgh gene fragments were then inserted into a bacterial plasmid. The plasmids were inserted back Fig 7: The experimental model into bacterial cells and the bacteria grown in culture. This is a quick and relatively easy way of getting large numbers of the plasmid vector. The plasmid vectors were isolated and then inserted into human breast cancer cells that are then grown in cell culture. Plasmids are small circular molecules of double stranded DNA that are found inside bacterial cells. Plasmids have the ability to be incorporated into the DNA of other cells so scientists use them as a way of inserting a gene into the DNA of another type of cell.. A second set of identical cells that did not had the hgh gene inserted are also grown in tissue culture. PCR and microarray technologies were then used to analyse and compare the gene expression between the two cell lines. (Fig 8.) Affymetrix Human Genome Array 6

Fig 8: Using microarray technology to analyse gene expression Reading a Microarray Red Spots Green Spots Yellow Spots Red spots mean that the gene was expressed more strongly in the hgh cells. The intensity of the colour of the spot gives an indication of the size of the increase in expression Green spots mean that the gene was expressed less strongly in the hgh cells. The intensity of the colour of the spot gives an indication of the size of the decrease in expression Yellow spots mean that the gene was expressed by both types of tissue i.e. there was no difference between the gene expression levels in the two types of cells. What did the scientists find? The Liggins Institute breast cancer study used a microarray analysis of 19,000 genes and found that a subset of 305 genes that were behaving remarkably differently when human growth hormone was secreted from the cells in the cell culture 5. The microarray results showed which genes were turned on or off in the cells that were secreting hgh compared to the cells that were not secreting hgh. Because the role of Fig 9: The microarray image produced from the Liggins Institute Human Growth Hormone Study some of the genes was known, the scientists could identify which part of the cell cycle was being disrupted. For example; if a gene that is known to play a role in making sure that damaged cells are destroyed is turned off, this helps in understanding why the cancer cells are reproducing. In addition to the genes that they knew the function of, some of the genes that were behaving differently had not been previously associated with cancer. Scientists are now studying these genes in the hope of finding their function in the cancer cell and improving their understanding of breast cancer. 7

Meeting Human Need Personalized treatments derived from genetic information The type of therapy used to treat a patient will depend on the type of cancer being treated and the stage of the disease. Traditional cancer treatments include: Surgery removal of the tumour Radiotherapy destruction of the tumour using ionising radiation Chemotherapy uses drugs which are effectively cellular poisons to target rapidly dividing cells or tissue that has low levels of oxygenation. Targeting rapidly dividing tissues causes problems as tissues such as the lining of the stomach are rapidly dividing and in traditional chemotherapy side affects such as an inability to hold down food and a loss of hair results from these broad target drugs. Some cancer tumours that are not well supplied with blood vessels and will have low levels of oxygenation they are hypoxic. Drugs have been designed that target hypoxic tissues. Recent Advances in Cancer Therapy In the last few decades there have been a number of advances in cancer therapy as a result of new technologies and improved genomic analysis of cancer. These include targeted therapies and the use of combinational therapies. In addition, individualised therapies hold great potential for the future. Targeted therapies: Targeted therapies are drugs that block the growth and spread of cancer by interfering with specific DNA or protein molecules involved in cancer. These therapies can be developed specifically to match the genetic and molecular characteristics of a patient s tumour. By targeting molecular and cellular changes that are specific to cancer, targeted therapies may be more effective than conventional treatments (such as chemotherapy) and less harmful to healthy cells. Tamoxifen was one of the first targeted therapies developed for breast cancer. The majority of breast cancers require the hormone estrogen to grow. Tamoxifen attaches to the estrogen receptor on the cell and stops estrogen from binding. When the estrogen receptors in the cell are blocked, the cell dies. Another example of a targeted therapy used in breast cancer is Herceptin. 25% of breast cancers have a high level of the Her2 receptor which leads to increased cell growth. Herceptin is an antibody specific for Her2 which blocks the function of this receptor by binding to it. This means that the cell is no longer getting the stimulus to divide and grow rapidly and will stop growing and most likely die. Combined approaches to cancer therapy: Depending on the type and stage of the cancer, combined therapy (which uses more than one treatment) can be more advantageous than using a single agent alone. This is due to a number of reasons. Some drugs may enhance the effectiveness of another when used in combination whereas some treatments may be more effective at different stages of cancer progression. In addition, the tumour may contain several sub populations of cells that are very similar, but not identical. Consequently, a single agent may not wipe out all the cancer cells, leaving some to repopulate the tumour. A combination of agents has more chance of killing all the tumour cells. Individualised therapy: No two cancers are exactly the same. By using molecular profiling doctors can identify those patients which are unlikely to benefit from a particular therapy, or who may suffer severe side effects from a particular treatment. 8

This can be achieved using technologies such as microarray and Real Time PCR. This development has come about through advances in technology, in particular, the human genome project, which has allowed scientists to look at genetic differences between individuals. It allows therapies to be tailored to an individual s needs. Using the example of Herceptin: If we give a patient with Her2 positive breast cancer Herceptin it has the potential to be an effective treatment. If we give Herceptin to a breast cancer patient who does not have high levels of the Her2 receptor, it will most likely have no affect at all. Potentially, by using microarray analysis, multiple genes can be analysed at once leading to improvements in the choice of therapies that are used. Using biotechnologies to understand cancer In order to treat cancer effectively, scientists need to know as much as possible about what is causing the cancer and the way the cancerous tissue behaves in different environments. They want to find out what increases the growth rate of the cancers and what can reduce the growth. This knowledge will improve our ability to find effective treatments for cancers. Scientists at the Liggins Institute in Auckland are investigating the role of growth hormone in breast cancer. Biotechnologies play a major role in this investigation and the consequent development of potential therapies. Technology Gene Profiling Genome Analysis How this technology has helped scientists understand of cancer About 5 10% of cancer patients inherit a genetic defect that gives them a susceptibility to cancer over their lifetimes. Many years of research using a number of biotechnologies went into achieving this understanding for example: PCR, DNA sequencing and gene mapping. Gene profiling allows scientists to identify individuals that carry specific alleles that increase their risk of developing cancer in their lifetime. Scientists have been able to study gene expression one gene at a time for many years using PCR technology. Primers specific to the gene of interest were used in the PCR mix to find out whether that gene was being expressed in the tissue. Microarrays are used to compare the expression levels of thousands of genes all at the same time, enabling scientists to study a genetic profile. The microarray technology can identify which specific genes a cell is using at a particular point in time. This means that we can compare which genes are turned on or off in different conditions (e.g. when cancer is present compared to when cancer is absent). The information from the microarray gives an overview of which genes are turned on or off, over or under expressed. To confirm how much more or less a gene is being expressed in a cancer cell line Real Time PCR technology is used. This is using PCR with primers specific to the gene as usual but a real time PCR allows scientists to further quantify the change in gene expression. Often the microarray analysis will identify genes that are already known to have a role in cancer. However, sometimes an experiment will identify a known gene which has not previously been associated with cancer. This is useful information as it identifies new targets for cancer therapy. 1. NZ Breast Cancer Foundation http://www.nzbcf.org.nz 2. Hanahan, D., Weinberg, R.A. (2000) The Hallmarks of Cancer Cell, Vol 100, 57 70 3. Perry, J.K., Emerald, B.S., Mertani, H.C., Lobie, P.E. (2006) The oncogenic potential of growth hormone, Growth Hormone & IGF Research 16 (2006) 277 289 4. Mukhina, S., Mertani, H.C., Guo, K., Lee, K.O., Gluckman, P.D., Lobie, P.E. (2004) Phenotypic conversion of human mammary carcinoma cells by autocrine human growth hormone, Proc Natl Acad Sci U S A 101, 15166 15171 5. X.Q. Xu, B.S. Emerald, E.L. Goh, N. Kannan, L.D. Miller, P.D. Gluckman, et al.,(2005) Gene expression profiling to identify oncogenic determinants of autocrine human growth hormone in human mammary carcinoma, J Biol Chem 280, 23987 24003. For further information contact Michal Denny m.denny@auckland.ac.nz; Jo Perry j.perry@auckland.ac.nz; Copyright Liggins Institute 2010 http://lens.auckland.ac.nz 9