Breast Cancer and Biotechnology Jacquie L. Bay, Jo K. Perry and Peter E. Lobie

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1 LENScience Senior Biology Seminar Series Breast Cancer and Biotechnology Jacquie L. Bay, Jo K. Perry and Peter E. 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. The number of people being diagnosed with breast cancer is increasing. In 1960, around 1 in 30 NZ women were diagnosed with breast cancer in their life time, today that figure is 1 in 9. That means that woman in NZ have an 11% chance of being affected by breast cancer. While the number of women being diagnosed with breast cancer is increasing, the death rate from breast cancer has decreased by 27% in the past decade 1. That is good news which is a result of increased understanding of the disease, improved screening, diagnostic methods, and treatment. Biotechnology has played a significant role in the development of knowledge of cancer and treatment of this disease. New Zealand has a large and active cancer research community that plays an important and often world leading role in cancer research. What is cancer? Cancer is cell growth out of control, causing the development of tumours which disrupt normal body function. Cancer may occur in any part of the body and 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 growth and reproduction in cells. Healthy cells have a limited life span and respond to signals which regulate cell growth and division. Cells will replicate a certain number of times and then go through programmed cell death (apoptosis), to be replaced by new cells. Unlike normal cells, cancer cells are immortal. They are not responding correctly the regulatory signals and will go on reproducing themselves over and over again. This fast rate of reproduction creates excess cells, forming tumours. The cell cycle is controlled by two families of molecules, Cyclin Dependent Kinases (CDKs) and Cyclins. Faults during the cell cycle such as inadequate growth and mistakes in the replication of the chromosomes will be picked up by these control agents and either the cell will be repaired, or the cell will self destruct (apoptosis). What makes a cell become cancerous? The transformation of normal cells into cancerous cells is the result of a succession of events, each giving the cell a growth advantage. That means an increased ability to grow, faster 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 and more aggressively. Cancers arise from changes in the cells. These may be changes in the DNA sequence (mutations) or they may be epigenetic changes that alter gene expression without altering the sequence of base pairs in the gene DNA methylation is one such epigenetic change that can arise as a result of changes in hormone exposure, or exposure to chemicals in the environment and in diet. (You will learn more about epigenetic changes in a later seminar Feast or Famine ). In those cancers that start with a mutation, these are usually arising from exposure to an environmental factor (a mutagen). In most cases, 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. 1

2 The Hallmarks of Cancer Scientists Douglas Hanahan and Robert Weinberg 2 have proposed that although there are multiple varieties of cancers, all cancers share six essential changes to the physiology of the cell that lead to the production of a successful tumour. They have proposed that these six acquired changes contribute collectively to the change from a normal cell to a cancerous cell. They called these six essential characteristics the Hallmarks of Cancer. 1. Uncontrolled Growth Normal cells grow or stop growing in response to signals start or stop. Cancer cells do not respond to these signals they will grow when there are no growth signals and will continue to grow when they are getting stop signals. So no matter what colour the traffic lights are they will go! Evading Death Angiogenesis Fig 2. The Hallmarks of Cancer (After Hanahan & Weinberg, 2000) Uncontrolled Growth Becoming Immortal Promoting Mutations Invading Tissues & Avoiding Detection 2. Evading Death Normal cells are meant to die when they get old or damaged. Signals in the cell start a process by which the cell membrane is disrupted and enzymes break down the cell contents, which are engulfed by neighbouring cells. This is called programmed cell death or apoptosis Cancer cells evade programmed cell death. They have the ability to repress the signals that start the process of apoptosis. 3. The ability to attract growth of new blood vessels (Angiogenesis) The oxygen and nutrients essential for cells to function are supplied by capillary blood vessels. Similarly, metabolic wastes (carbon dioxide, urea and water) are removed via the blood vessels. This means that all cells must be in close proximity (within 100 m) 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. 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. Each time a cell divides, the telomeric DNA get slightly shorter. When it gets so short that it cannot protect the chromosome the cell dies. In cancer cells the temoreric DNA does not get shorter because an enzyme called telomerase is released which lengthens the telomeres so the cell can keep on replicating endlessly. 5. Invading other tissues and avoiding detection 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. Normal Cell Uncontrolled Growth Capillary Invasion Angiogenesis Cancer Cells Poor Prognosis Metastasis Invasive Cancer Cell Fig 3. Cancer Progression 2

3 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. Cell Culture: Making & culturing transgenic cell lines Human cells, including cancer cells, can be cultured outside the human body in plastic flasks kept in incubators. 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. Fig 4: Cell culture flasks Fig 5: PCR and Gel Electrophoresis are used to study the expression of genes in individual tumour samples. These gel photos show Beta-Actin, a house keeping gene that is used as a loading control and two genes of interest hgh and the cell surface receptor for hgh, hghr. 3

4 Breast Cancer and Human Growth Hormone Human growth hormone (hgh) is made and secreted by the pituitary gland 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. Acromegaly is another condition caused by prolonged overproduction of growth hormone in adults. It results in excess growth of the bones in the lower face, hands and feet. Richard Kiel, the actor made famous in his role as Jaws in the James Bond movies The Spy Who Loved and Moonraker, suffers from this condition. In addition to the hgh produced and secreted from the pituitary gland, hgh is also produced and secreted locally in different sites around the body. These sites include localised 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 6). Fig 6. Excretion of Growth Hormone in Humans Studies conducted at Auckland University s Liggins Institute 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 and benign tumours. Scientists used tissue culture to study the effect of differing levels of hgh on the development of breast cancer cells. They have found that when hgh is secreted from breast cancer cells (autocrine hgh) it increases cell growth rates and the cells are more invasive but when hgh is added to the tissue culture as if it had been secreted from the pituitary gland (exogenous hgh) into the blood stream, it does not increase cell growth as effectively 4. The scientists used this difference to find out about the molecules that are causing the breast tissue to become cancerous. Microarray and PCR technologies were used to try to find out what was causing the difference. Fig 8: Autocrine hgh production in cells increases growth. Control: no hgh Cells producing hgh Fig 7: The control cells 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. Gene expression what is going on to create this phenotype? In studying disease, scientists want to find out which genes are expressed (turned on) in different situations. 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 PCR technology. Microarrays are exciting 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). 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 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 some of the genes was known, the scientists could identify which part of the cell cycle was being disrupted. E.g. 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. 4

5 Affymetrix Human Genome Array The experimental model Fig 9: The experimental model The hgh gene is isolated and cloned into a plasmid vector The vector is inserted into human breast cells that are then grown in cell culture A second set of identical cells that have not had the hgh gene inserted are also grown in tissue culture PCR and microarray technologies are used to analyse and compare gene expression between the two cell lines. Microarray technology is used to scan 19,000 genes to look for differences in gene expression Fig 10: 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 is on the microarray chip? A microarray is a small glass slide that contains tiny fragments of known DNA sequences in different spots on a slide. A Human Genome microarray will contain small fragments of each of the genes in the genome. They are made robotically, with DNA probes being attached on vertical stacks onto the glass slide. Each spot on the slide contains multiple copies of the same probe. When the 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 using computer technology. Using PCR Technologies PCR the polymerase chain reaction, amplifies DNA and is used in multiple ways. In gene expression studies PCR is used to amplify specific fragments of a gene to find out whether that gene is being expressed in the tissue being studied. RNA is extracted from the cells Reverse Transcriptase reaction produces cdna from the RNA The Polymerase Chain Reaction (PCR) using specific primers produces multiple copies of the target DNA Fig 11: The microarray image produced from the Liggins Institute Human Growth Hormone Study 5 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.

6 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. 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. 1. NZ Breast Cancer Foundation 2. Hanahan, D., Weinberg, R.A. (2000) The Hallmarks of Cancer Cell, Vol 100, 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) 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, 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, For further information contact Jacquie Bay, j.bay@auckland.ac.nz; Jo Perry j.perry@auckland.ac.nz; Peter Lobie p.lobie@auckland.ac.nz Copyright Liggins Education Network for Science, 2008 The Liggins Education Network for Science Bringing Schools and Scientists Together 6