CANCER BY SIR. SEMWEZI ENOCK Cancer affects many people in many different ways. You may be a carrier or a sufferer wanting to know more about how and why the disease occurs Cancer results from the uncontrolled proliferation of cells in certain tissues in the human body. The onset of cancer can be triggered by: Long term exposure to certain viral infections Chemical carcinogens In a small minority of people, by an inherent mis-wiring in the cells, All of which can destabilize the delicate balance that controls how a cell Divides (proliferation), Changes into new cells (differentiation) How a cell dies (apoptosis). NB: Cancer cannot spread from one person to another (like the common cold for example, which is spread by viruses). Cancers arise from cells within our own bodies. DID YOU KNOW THAT? That normal cell are transformed into cancers as a result of changes in these genetic networks at the molecular, biochemical and cellular level. Almost all known animal species on the planet can develop cancer. The only species that we know of, that is highly resistant to developing cancer, is an extraordinary rodent called the naked mole rat. These tiny rodents (each one weighs less than 35g), also happen to have an exceptionally long life (about 28 years). They have a special type of sugar called hmm-ha (high molecular mass -hyaluronan). This glue-like molecule make the naked mole rat cells particularly sticky, preventing cell overcrowding and forming tumours. What is cancer? Cancer is a disease that involves changes or mutations in the cell genome. These changes (DNA mutations) produce proteins that disrupt the delicate cellular balance between cell division and quiescence, resulting in cells that keep dividing to form cancers. Already known causes of cancer include: cigarette smoking is associated with a high risk of developing lung cancers.( hydrocarbons again in the tar) age, almost 60% of all cancers that you see around the world is linked to age. And this is found typically in people over the age of 65. So this is a very clear cut evidence that the longer you live, there's an increased risk of getting cancer obesity, lack of physical activity, high calorie diets, all of which increases the risk of getting certain types of cancers. Viruses can be a big role causing cancer;e.g. Cervical cancers. Where the infection with a virus called as the Human Papilloma Virus has shown to be a causal agent for developing cervical cancers in women. The good news is that It's not simply one single infection with HPV that gives you the cervical cancer. You need a lot of hosts of contributing factors and that includes things like a long sexual history with the different partners, diet, life style, smoking, all of which can increase the risk of getting cervical cancers in women who have been infected with HPV. GENES The gene, the genome and you A gene is a section of DNA that, once transcribed and then translated, codes for a protein. DNA stands for DeoxyriboNucleic Acid. The genome is all of the DNA in a cell that specifies the form and function of an individual organism. Most cells contain a complete genome in the form of multiple, linear DNA molecules, compacted into chromosomes. A gene is a subset of the genome that is switched on (ie expressed) by the cell to make mainly proteins, and a cell can make hundreds of proteins through more than 20,000 genes. Proteins are the building blocks of a cell. A properly functioning cell is controlled by genes, which produce the correct subset of proteins, at the correct times, in the correct amounts, and in the correct places. Some proteins also act as switches, turning genes on or off, which in turn can dictate how a cell behaves. Correct gene expression is essential for normal cellular function. Cell growth and division is part of normal function. What is epigenetics and why is it important in cancer? it is now described as a mechanism through which the expression of a gene can be controlled without affecting the sequence of DNA. Rather, epigenetics controls the packaging of DNA and how it is accessible for gene expression. Epigenetics controls the expression of genes. This is done either by: turning the gene on or off by simply adding a chemical tag or mark on DNA (such as a single methyl group to a cytosine) or controlling how DNA can be unwrapped to enable access to key genes. Packaging DNA into structures called chromatin is important for compressing the massive human DNA inside a micrometer-sized cell. In order for a gene buried deep inside this chromatin to be expressed, DNA needs to unwrap first to allow access by the synthesis machinery. Specialised proteins control the wrapping and unwrapping of DNA and genes. Epigenetics is key to normal cellular processes, for example, during development, through the inactivation of one of the two X-chromosomes in females or ensuring that the same chemical marks / tags on the DNA are passed on from the mother or father to the offspring (imprinting) or during cell division. Exciting recent research also shows that epigenetic marks can arise from environmental factors including diet and smoking. To summarise, the genome contains the DNA sequences which form genes, whereas the epigenome includes chemical modifications on the DNA itself and/or histone proteins, which affect DNA packaging and gene expression. epigenetics can also alter the expression of genes resulting in the growth of cancer cells. Epigenetic silencing of cancerrelated genes occur in a number of different ways. Genes may be silenced by the addition of chemical marks (like methyl groups) in the promoter region of a tumour suppressor gene. A promoter is a region of DNA located in front of any gene and which controls gene expression. Therefore, if the promoter of a tumour suppressor gene is switched off by the chemical mark, the protein will not be made and therefore the brakes on cell division is removed. Alternatively, the tumour suppressor genes may be compacted tightly within the chromatin, preventing them from being expressed. A potential avenue for cancer therapy would be to enable access to these tumour suppressor genes, by inhibiting enzymes that wrap or compress DNA. Some examples of successful inhibitors of these DNA compaction enzymes are azacitidine, an inhibitor of DNA methylation, and eninostat, an inhibitor of histone deacetylation. Used in combination this treatment prevents DNA methylation from occurring and also prevents chromatin compaction, therefore ensuring that genes can be expressed. The efficacy of this treatment was shown recently in lung cancers where patients showed a total response following a one year course with combined epigenetic therapy of azacitidine with eninostat. DNA mutations and rogue cells Cancer is a heterogenous disease, involving an accumulation of multiple mutations in a multi-step process. Tumours are not simply a group of cells that divide uncontrollably, but a complex tissue with different cell types all of which actively work together. DNA mutations result in defects in the regulatory circuits of a cell which disrupt normal cell proliferation behaviour. However, the complexity of this disease is not as simple at the cellular and molecular level. Individual cell behaviour is not autonomous and usually relies on external signals from surrounding cells in the tissue or microenvironment. There are more than 100 distinct types of cancers and any specific organ can contain tumours of more than one subtype. This provokes several questions. 1. How many of these regulatory circuits need to be broken to transform a normal cell into a cancerous one? 2. Is there a common regulatory circuit that is broken among different types of cancers? 3. Which of these circuits are broken inside a cell and which of these are linked to external signals from neighbouring cells in the tissue? The answers to these questions can be summarised in a heterotypic model, simplified down to a few fundamental concepts that alter the internal cell circuit and cell physiology, that was first proposed by Douglas Hanahan and Robert Weinberg. These fundamental concepts or Roads to Cancer will be covered over the next two weeks. This currently accepted model can be best described using a traffic light analogy. Produce ‘Go’ signals: Normal cells carefully control how and when they divide. This control is critical to maintain the normal tissue structure and function. Cancer cells, on the other hand, disrupt this control by producing and releasing their own chemical signals (or GO proteins) that keep the cell division ‘switch’ on for much longer. Examples of these ‘Go’ proteins are growth factors that will bind to proteins on the surface called receptors and will allow continued cell proliferation (covered in more detail in Step 2.4). Override ‘Stop’ signals: Again, cell division in normal cells is usually kept in check by proteins that put the ‘brakes on’, so called growth suppressors or Tumour Suppressor Proteins. Cancer cells cleverly bypass these brakes, or deactivate these STOP proteins, in order to continue dividing (Step 2.4). Defying death: Cell death is part of life in normal tissues. A normal cell will be told to kill itself if the cell division conditions are not ideal, or if the cell has sustained excessive damage to its DNA (due to exposure to DNA damaging agents) or simply if there is too much or inappropriate cell division. Cancer cells however, produce proteins that sneak past or knock out these death-inducing proteins and allow them to grow and divide (Step 2.7). Keeping the supply lines open: Angiogenesis means stimulating new blood vessel growth. Cancer cells will not be able to sustain their own growth without their blood supply (blood vessels that carry oxygen and nutrients). Therefore, cancer cells secrete factors that stimulate new blood vessels to expand alongside their own cell division (Step 3.2). Spreading out – Metastasis: This is the final stage in which cancer breaks through the tissue barrier (stroma) and spread through the bloodstream and lymphatic system to colonise tissues in remote sites. A vast majority of death in cancer is due to metastasis (Step 3.4). Others: A host of new concepts have also been implicated in cancer development and progression. This include the idea of the cancer stem cell (CSC). This concept posits that cancer arises due to disruptions in differentiation. Differentiation is the process in which a stem cell is instructed to change or differentiate into a specialised cell such as a heart cell, liver cell, neuron etc. An increasing number of tumours now show subpopulations of CSCs, adding weight to the idea that cancers arise due to disrupted differentiation signals Cell signalling, oncogenes and tumour suppressors Chemical signals and cancer cells Cell signalling involves the release from one cell of a chemical signal that is recognised by other cells, usually because the recipient cell possesses a receptor that is highly specific for the signal molecule. Signalling, or cell communication, is required to co-ordinate cell behaviour and is particularly important in organisms made up of many tissues and cell types. Multicellular organisms depend on cell signalling for correct development, tissue maintenance and homeostasis. Signals may instruct cells to survive or to die (through programmed cell death or apoptosis), to differentiate and become a specialised mature cell type, or to divide (proliferate). These instructions are part of the normal processes of development and tissue renewal (eg in skin or gut), and can each go wrong in cancer. Signal-receptor interaction starts a chain of molecular changes within the cell. The molecular relay or intracellular signalling pathway allows amplification of the signal and dispersal of the signal to different parts of the cell to bring about changes in cell behaviour. Tight control is needed over cell division (proliferation) to maintain tissues of the correct size and containing the correct cell types to perform the normal functions of that tissue. Dividing cells are said to go through the cell division cycle. To begin, the cell cycle signals are needed that promote the transition from the resting phase, G1, to begin the synthesis of DNA in S phase (when each of the chromosomes are copied in preparation for cell division). In cancers, one or more of the signalling components becomes activated inappropriately, usually as a result of gene mutations. The mutated genes are known as oncogenes, which produce hyperactive forms of signalling proteins that can promote cell division in the absence of the correct signal. Tumour suppressor genes normally function to stop cells from beginning the cell cycle. They produce cell cycle regulators that usually function to keep the cell in check until the correct signals for cell proliferation are received. Mutations in tumour suppressor genes also occur in cancer cells, which can then more easily begin the cell cycle without the proper instructions. Cancer cells escape the normal controls over cell survival, proliferation and organisation. Cancers go through a series of different stages as cancer cells acquire successive mutations in oncogenes and tumour suppressor genes. Excess proliferation of cells within their original tissue can lead to the formation of a benign tumour. Accumulation of further mutations in key genes can lead to cells invading surrounding tissues, when the tumour is said to become malignant. Malignant tumours can cause damage that may be life-threatening, including spreading to other parts of the body in a process termed metastasis. These events occur due to a series of mutations in genes that normally regulate cell proliferation, survival and organisation within tissues. How cancer cells defy death Death is a part of life, and this is also the case in tumours. By the 1960s, we knew that spontaneous loss of tumour cells was part of the process of growth of tumours. In 1972, three scientists (Kerr, Wylie and Currie) proposed that cancer occurs not simply because of excessive cell proliferation, but also due to reduced cell death. In the first session this week, we explored some of the roads to cancer and how normal cells will kill themselves rather than tolerating irreparable damage to their DNA. We shall now examine how cancer cells defy death. Before we do this, I would first like to explain how cells die. There are two common ways this can happen: 1. Necrosis: This is a really messy way for a cell to die and typically happens to cells in injured tissues (untreated wounds, frostbite, chronic infections etc). The cells swell up and burst, spilling all the contents outside. This leads to an inflammatory response and the process is uncontrolled. If left untreated, necrotic cells can lead to a build up of decomposing tissues, in turn leading to gangrene. 2. Genetically-controlled Programmed Cell Death (PCD): Among the most common form of PCD is apoptosis (from the Greek for falling leaves). This is a highly-controlled operation, dictated with almost military-precision by genes. No messy spilling, no inflammatory response, the cell simply dismantles itself systematically, like taking down the scaffolding from a building. The DNA and cellular components are packaged neatly into little bodies called ‘apoptosomes’. All evidence that a cell ever existed in that location is removed completely AND (this is fascinating bit) this is usually done by a neighbouring cell or by an immune cell called a phagocyte, which eats up this dying apoptotic cell. Another form of PCD that is getting a lot of attention is autophagy (literally eat oneself). This is the typical response of a starving cell trying to protect itself by degrading some of its internal proteins by organelles called lysosomes. So now that we have looked at ways in which cells die, lets look at how tumours develop due to reduced cell death. One of main pathways that is disrupted in cancer cells is apoptosis. Apoptosis is normal. Hundreds of cells in our body die regularly due to apoptosis, eg during development. Did you know we were all born as ducks? We all had webbed fingers early in our development in the womb. Apoptosis instructs the cells in the webbing of our fingers to kill themselves, and that is how our fingers are sculpted into digits! The same apoptotic process also happens in fine-tuning our immune system or in getting rid of the unfertilised egg during the menstrual cycle. Controlling the process of apoptosis is critical and any disruption can lead to either: Too much apoptosis (like when brain neurons die in Alzheimer’s disease), or Too little apoptosis, as we find in tumour development and metastasis or spread. Apoptosis is hard-wired into every cell, like a ticking time bomb, only switched on by gatekeeper or guardian proteins called tumour suppressor proteins (such as p53 or retinoblastoma, which we covered in the Step 2.4). In cancer cells, these tumour suppressor proteins cannot do their job because of mutations or inhibitors (such as viral oncoproteins) prevent apoptosis being switched on and the cells in the tumour will continue to grow. If you are interested, further information about the specific genes and proteins and the pathways involved are provided in the links below. In the next session, we will look at other ways that tumours develop and grow. Angiogenesis: how cancer cells switch on the blood supply One of the main pathways to cancer development is angiogenesis. Angiogenesis can be defined as the formation of new capillaries from pre-existing blood vessels by migration and proliferation of specialised cells, called endothelial cells, which form the inner wall of the blood capillary. It is a key step in a number of physiological processes such as wound healing or the female menstrual cycle. In tumours however, angiogenesis is responsible for the growth of the tumour, invasion and spread (metastasis). How do tumours switch on angiogenesis? Like all normal tissues, tumours are also dependent on oxygen and nutrients provided by the blood vessels (capillary microvasculature). However, endothelial cells do not normally proliferate and therefore, initially, the growth of the tumour is restricted (usually no more than a few mm3 in size). In the later stages, with subsequent mutations, tumour cells release proteins (called angiogenic factors) that stimulate the proliferation of endothelial cells to create a new vasculature of their own. This tumour neo-vasculature triggers a rapid expansion and growth of tumour cells. In most normal tissues, proliferation of endothelial cells is tightly regulated through the production of naturally occurring pro-angiogenic factors (such as VEGF – vascular endothelial growth factor) and angiogenic inhibitors (such as endostatin). The levels of angiogenic inhibitors predominate, thereby keeping blood vessel growth in check. Consequently, the structure of vasculature in healthy tissues is regular and fed by straight vessels that branch into smaller capillaries and microvessels. Tumours flip this angiogenic switch by releasing angiogenic factors, which results in the stimulation of angiogenesis. This neo-vasculature in tumours is disorganised in every aspect of their structure and function. They are tangled, irregular, oversized, leaky and with areas that may lack these microvessels altogether. The blood flow may be brisk in one part of the tumour but static in another, or flow in reversible directions. The major consequences of this erratic vasculature are: Uneven drug delivery, which results in either the drugs being ineffective or the tumour cells becoming resistant to the effectiveness of the drug It increases the chance of the cancer cells spreading to other locations (metastasis). What are the molecular mechanisms underlying angiogenesis in tumours? Angiogenic factors released by tumours, such as vascular endothelial growth factor (VEGF), activate endothelial cells. This results in the secretion and activation of a special class of enzymes that breaks down the surrounding tissue protein matrix, called matrix metalloproteinases (MMPs). MMPs break down the thin outer lining of the blood vessel, called the basement membrane. The endothelial cells of the blood vessels are now free to migrate towards the tumour cells and proliferate into the space created by the action of the MMPs. Another class of receptors, called integrins, that are found on the surface of the endothelial cells, act as the ‘velcro’ by pulling the cells towards tumour cells. These newly formed microvessels secrete further growth factors, such as platelet-derived growth factor (PDGF), which attract supporting cells and the eventual expansion of the capillary network. Anti-angiogenic targets for treating cancer Drugs targeted at inhibiting angiogenesis have had some success in Clinical Trials. Avastin® (bevacizumab), which neutralises the angiogenic factor VEGF, was shown to be effective against recurrent glioblastoma (a type of brain tumour). Sutent® blocks the receptor for VEGF. However, some cancers have shown to be unresponsive to anti- angiogenic drugs; Avastin® was ineffective against colon cancers. Key concepts Angiogenesis is the formation of new blood vessels. This process is activated by cancer cells to enable tumours to grow in size In most tissues, angiogenesis is switched off by large amounts of anti-angiogenic proteins The vascular network in tumours is irregular, oversized and leaky, resulting in increased risk of spread and developing resistance to drugs Cancer cells release angiogenic factors which stimulate blood vessels to divide and expand to form vascular networks Anti-angiogenic drugs target these factors to stop expansion of blood vessels. Metastasis: when cancer cells spread What is metastasis? Metastasis is the spread of cancer cells throughout the body. It is the most common cause of death from cancer but it still remains one of the most poorly understood areas of cancer research. Most solid tumours are either benign or malignant. Benign tumours can grow and have all the features of cancer we have highlighted so far butthey are always contained within the tissue basement membrane, with defined shapes, whatever their size. Malignant tumours (cancers), by contrast, break through the basement membrane barrier and pass into the blood vessels and metastasise. Cancer invasiveness represents one of the first stages of metastasis, which is the spread of cancer cells throughout the body and the colonisation of distinct, often distant tissues. The metastatic process Metastasis is an inefficient process, which can be broadly divided into the following 6 stages: 1. Localised invasion: This involves the growth of the primary tumour and breaking out through the basement membrane. 2. Intravasation: Tumour cells squeeze through the capillary wall and into the blood and lymph. A vast majority of tumour cells do not survive this harsh environment because of their interaction with the blood components, including lymphocytes of the immune system. 3. Transport through circulation and arrest in microvessels of secondary organs: Tumour cells that do survive now accumulate in areas of sluggish circulation in the body, such as the microvessels in the lungs, liver etc. This is also one of the reasons why heart cancers are rare. 4. Extravasation: Tumour cells squeeze back out of the blood vessel into the surrounding tissue. 5. Formation of micrometastasis: The process of growth and proliferation, without the induction of angiogenesis 6. Formation of macrometastasis: The last stage of actively growing and angiogenic tumour. Studies have shown that after extravasation, a subset of tumour cells may remain either as: dormant, solitary cells dormant, pre-angiogenic micrometastasis or actively growing, angiogenic metastasis. It is usually the active, angiogenic metastatic tumour that is clinically detectable with imaging. The molecular genetics of metastasis Metastasis is a complex process controlled by molecular genetic pathways that are either inherent in the tumour cell or dependent on a more complex interaction between these tumour cells and cells from the host tissue/organ (tumour– host interaction). The inherent genetic pathway underlying metastasis is often due to the abnormal expression of genes that are not essential, but are usually switched on during stress conditions. The genes themselves remain typically unchanged, but the levels of the protein that they express usually change. In order for a cancer cell to metastasise, it must have two essential functions: 1. Invasiveness, which allows tumour cells to break through the tissue basement membrane of the main tumour, surviving the harsh environment of the blood and lymph, reach the capillary bed of the target organ and form a secondary metastasis at the second site. The key molecular genetic switch needed for invasion is EMT (epithelial-mesenchymal transition), wherein tumour cells switch on the expression of proteins needed for this role. They reduce proteins that enable them to stick to the matrix (eg E-cadherin) and overproduce proteins that enable degradation of the matrix (MMPs) and motility (integrins). You may recall these proteins from the earlier session on angiogenesis. Intriguingly, this switch is reversed after the cancer cells have reached the secondary target tissue, called MET (mesenchymal-epithelial transition). This reversal enable cancer cells to now colonise the secondary site and form micro- and macro-metastatic tumours. 2. Anchorage-independent survival enables tumour cells to survive the harsh environment in the blood and lymph, without the need of a scaffold to hold on to. This is in contrast to normal cells, which usually die from a unique type of cell death called anoikis when they lose contact with their surrounding matrix. Genetic programs that confer these properties of invasiveness and anchorage-independence are absent in benign tumour cells. The cancer stem cell and other genetic pathways to cancer Most human cancers are complex tissues arising from the clonal selection of subpopulations of cells with distinctive genetic heterogeneity. As we have seen from the clonal origins model, most tumours show varying degrees of differentiation, mutations, vasculature or metastatic potential. Recent research suggests an alternative, but not mutually exclusive, explanation for this tissue heterogeneity, termed cancer stem cell (CSC) theory. This is based on the observation that, in some tumours but not all, cells show abnormal differentiation. The diverse subpopulations of cells in tumours represent cells at different stages of differentiation. Generally, differentiated cells in adult organs have a finite lifespan, ranging from days (intestine and skin), months (red blood cells) to decades (neurons and memory-type lymphocytes). Dead cells are replaced by adult stem cells which have two essential properties, self-renewal and differentiation. Remarkably, an adult stem cell can do both in one go, through asymmetrical cell division. When an adult stem cell divides to form two daughter cells, one of them retains all the stem cell properties (self-renewal) while the other is committed to differentiate into a specialised cell (such as a liver or heart cell). Adult stem cells tend to divide very rarely, in stark contrast to differentiated cells which divide rapidly (called progenitor cells or transit-amplifying cells). The rapid division of the progenitor cells is soon exhausted and the cells become terminally differentiated (ie do not divide but perform specialist functions, such as a neuron). CSCs arise due to the oncogenic transformation of normal adult stem cells in any of these stages. Initially implicated in metastatic blood cancers, CSCs have now been also implicated in certain types of breast and colon cancers. Other genetic pathways The genetic pathways of cancer development listed below are based on some of the recent research in the area but are yet to be fully validated and accepted by the global research community. Hence, these are categorised as emerging pathways: 1. Evading the immune system: Almost all cancers show the presence of cells of the immune system at varying densities. Their presence suggests an antitumour response by the immune system to eliminate the aberrant cells. However, there is compelling evidence which suggests that, paradoxically, the inflammatory response by the immune system exacerbates cancer progression, by supplying molecular mediators (such as growth factors, angiogenic and invasive factors) which increase proliferation, growth and spread of tumours. 2. Genetic reprogramming of cellular metabolism: The uncontrolled cell division that defines cancer also poses a huge demand for energy resources to fuel this growth. Otto Warburg was the first to notice that cancer cells reprogram their glucose metabolism pathway to sustain the rapid cell proliferation. Glucose is broken down (glycolysis) even when oxygen levels are low (aerobic glycolysis). One of the ways cancer cells do this is by increasing the number of glucose transporters (Glut1) which import glucose into the cell. 3. Immortality of cell division: Another defining feature of cancer cells is their ability to divide endlessly, unlike normal cells, which undergo senescence (stop dividing). An internal clocking device, the telomeres (hexameric DNA repeat sequences, which protect the ends of chromosomes) determines how many times a cell can divide, but usually gets shorter every time a cell divides. Telomerase, the enzyme that adds telomeres, is usually absent in normal cells but is functionally active in more than 90% of immortal cancer cells. To summarise, some of the key molecular genetic pathways to cancer development involve oncogenes that maintain cell division, dysfunctional tumour suppressor genes, antiapoptotic genes that resist cell death, pro-angiogenic genes, and genes that activate invasiveness and metastasis. Emerging pathways include altered energy metabolism, genome instability and inflammation. A broad understanding of these key genetic pathways will help further our research into treating human cancer.