Research Overview

Molecular Genetics of Cancer Division

A cancer develops when one cell in any tissue escapes normal control processes and multiplies rampantly, eventually overcoming all of the body's defences. This Division is exploring how cancers arise by studying how accidental damage to genes that control cell production leads to cancer. For instance, certain genes normally limit the cell numbers in our tissues by ordering any damaged or superfluous cells to die. This healthy and essential process of programmed cell death is called "apoptosis". In fact, every second of our lives, about a million new cells are born and a million old or damaged cells must die. This balance is essential for health. Too much cell death may cause degenerative diseases such as Alzheimer's or Parkinson's. Conversely, too little cell death results in cell accumulation that may start a tumour. We have identified several of the genes that control apoptosis and determined how their alteration causes tumours. Eventually, understanding of apoptosis regulation should lead to new forms of cancer therapy.

Background

Over the past two decades it has become increasingly clear that cancer is produced by errors in the normal genetic programs that guide the multiplication, specialisation and death of our cells. These errors reflect damage to critical genes (see Cancer: a disease provoked by damaged genes).

The focus of research in our Division is to clarify the relevant genetic programs and to establish how they are altered during development of a tumour. We attempt not only to identify the key regulatory molecules and also to establish their natural role, by modifying the genes that specify them, in mice.

For normal bodily function, the birth of new cells must be balanced by the death of others. Throughout life, redundant, effete, damaged or infected cells must be eliminated. Cells die by an intricate suicidal process called apoptosis (see Apoptosis - a matter of life or death).

Inhibition of apoptosis plays a central role in the development of many cancers. Excessive apoptosis, on the other hand, underlies many autoimmune and degenerative diseases. We are therefore studying how apoptosis is normally kept under control.

Clarifying the biochemical pathways controlling cell death will inevitably have clinical benefits, by suggesting novel strategies for inducing cell death in cancer cells, or for preventing cell death in degenerative disorders.

Cancer: a disease provoked by damaged genes

Cancer has been called a "malady of the genes". A tumour results from the inexorable multiplication of a single cell in the body, triggered by inadvertant damage to critical genes within that cell.

The now accepted "multiple hit" theory of cancer suggests that, as people age, their cells accumulate random damage to their genes (mutations). By chance, some of the damage will occur in the critical genes that normally tightly control the proliferation and survival of cells. If any cell accumulates sufficient "hits" to destroy all the genetic brakes holding it in check, that single cell can grow out of control and found a dynasty of "rogue" cells - a cancer.

Cancer is predominantly a disease of aging but paradoxically also occurs in young children. Most cancers of the young originate in inherited mutations within this same group of genes. The difference often is that the child has inherited a single mutation from a parent, greatly increasing the risk that further accidental mutations will trigger cancer early in life. However, only a small minority of cancers are thought to arise from inherited mutations. Instead, most tumours develop due to random mutations within the various cells of the body - a cruel lottery.

Some cancer-provoking (oncogenic) mutations knock out or inactivate critical genes. Others provoke inappropriate gene activity. When a cell has accumulated a critical load of oncogenic mutations (perhaps six to ten), it begins to divide uncontrollably, eventually threatening the life of the individual.

Cancer researchers have uncovered a class of genes called proto-oncogenes that are key players in regulating normal cell growth and division. Mutation of a proto-oncogene can turn it into an oncogene - a gene capable of causing the host cell to become cancerous.

Researchers have also discovered another, very different class of genes, called tumour suppressors. The products of these genes serve as sentinels inside the cell, constantly monitoring it for hazardous mutations; if a cell has acquired such a mutation, the tumour suppressor springs into action to eliminate the cell before it can cause trouble.

Unfortunately, tumour suppressor genes, like all other genes, are themselves vulnerable to mutation. Their loss or inactivation is a common prelude to cancer, and consequently many different cancers contain mutated tumour suppressor genes. For example, the best known tumour suppressor gene, p53, which cancer researchers have dubbed "Guardian of the Genome", is damaged in over half of all tumours. In the specific case of lung cancer, it is known that chemicals from tobacco smoke can damage the p53 gene in lung cells and thereby set them on the path to malignant growth.

A third class of cancer-provoking gene has recently been recognised: genes that frustrate an in-built cell suicide program known as apoptosis. Cells that are incapable of dying when they should are as dangerous to the organism as cells that divide too often. Apoptosis has become one of the most exciting areas in cancer research, and is currently the principal focus of researchers within the Molecular Genetics of Cancer Division.

Apoptosis - a matter of life or death

In many tissues of the body, such as the gut or the blood, cells are continually produced throughout life. Therefore any cells that are damaged as well as those that are no longer needed must be eliminated. To ensure their removal, cells have developed an in-built suicide program now called apoptosis.

The term derives from a Greek word used to describe the inevitable leaf-fall from deciduous trees, which occurs in a "programmed" fashion every autumn. An Australian, John Kerr, and two Scots, Andrew Wyllie and Alistair Currie coined the term to describe the normal process by which cells die. Kerr realised from his electron microscope studies that cells dying of 'natural causes' did so in a very predictable, reproducible fashion. He and his colleagues hypothesised that there was an inbuilt (programmed) process - hence the terms apoptosis or programmed cell death.

During apoptosis, the chromatin in the nucleus of the cell condenses and its DNA becomes fragmented. At the same time, the cells shrink and the membranes start to bleb, forming membrane-enclosed vesicles called apoptotic bodies. These cellular relics are then tidily packaged as small vesicles and consumed by neighbouring cells. Once started, the process is over within minutes.

Apoptosis is essential for sculpting the developing embryo and maintaining the proper number of cells in the tissues and organs of the adult. For example, early in the development of a human embryo, the fingers and toes are conspicuously webbed but, at the proper time, the unwanted cells are eliminated by apoptosis. (In contrast, this cell death program is not activated in ducks, who retain their webbed feet, to great benefit!)

Cell death is also of great importance in the development of the brain. Explosive growth in the embryonic brain initially creates many more nerve cells than the newborn needs. Once the brain has wired itself for action, however, the surplus millions of nerve cells commit harikari. If this process is activated inappropriately later in life, neurodegenerative disease may ensue.

Apoptosis is essential to control cell numbers in the immune system. During an infection, lymphocyte numbers increase dramatically to fight the invading foe. The lymphocytes kill infected cells by inducing them to die by apoptosis - murder by autosuggestion! Once the battle is won, however, lymphocyte numbers must subside to normal levels. So the soldier cells then fall on their own swords - altruistic suicide!

bcl-2: an oncogene that frustrates apoptosis

The first direct connection between cancer and genes controlling apoptosis was made here, in 1988, by Dr David Vaux, then a PhD student in the Division with Professors Jerry Adams and Suzanne Cory. They were studying bcl-2, which had been recently identified as the gene affected by a chromosome abnormality that hallmarks the most common human lymphoma (follicular lymphoma). The new gene seemed likely to be a gene that can contribute to cancer development (a proto-oncogene), but its function in normal cells was totally unknown.

To explore bcl-2 function, Vaux introduced an activated bcl-2 gene into cells whose ability to grow in the 'test tube' normally requires a special growth factor or cytokine. He found, most unexpectedly, that this dependency had now been broken. Although the cells remained unable to divide and multiply in the absence of the cytokine, they survived perfectly well for many days, and would start proliferating again when cytokine was added back to the culture dish. This now-classic experiment demonstrated that bcl-2's job is to enhance cell survival by blocking the apoptosis program.

How does the bcl-2 gene in a follicular lymphoma cell differ from that in normal cells? The B lymphocyte which gave rise to the tumour happened to incur breaks in its genetic material at both the bcl-2 gene on chromosome 18 and at an antibody gene on chromosome 14. (Transient breaks at the chromosome 14 site normally occur in B lymphocytes to create a functional antibody gene). DNA-splicing enzymes usually repair such breaks but, in this cell, they accidentally joined the wrong chromosome ends together, thereby grafting the bcl-2 gene to a turbo-charged antibody gene. The result was that the cell over-produced the life-prolonging Bcl-2 protein. Thus, when the time came for the B cell to die, it had acquired a dangerous new lease on life and the stage was set for cancer.

bcl-2 and lymphoma development

Together with Dr Alan Harris, researchers went on to transplant the mutant bcl-2 gene into eggs of mice, thereby creating new strains of mice in which Bcl-2 protein is over-expressed in all lymphocytes.

These 'bcl-2 mice' accumulated vast numbers of lymphocytes in their blood, spleen and lymph nodes. Dr Andreas Strasser investigated these cells and showed that they persisted even in the absence of vital cytokines. Significantly, they also survive otherwise lethal doses of radiation and cytotoxic drugs. Thus, a surfeit of Bcl-2 can frustrate life-saving therapy.

Very significantly, a proportion of the mice went on to develop lymphoma. It thus became clear that the relationship between the bcl-2 chromosome translocation and follicular lymphoma was no accident. The animal model had proved beyond any shadow of doubt that perturbing the bcl-2 gene was indeed the root cause of this malignancy.

The tumour-prone bcl-2 mice provided further clues about the cancer process. Not all of them developed tumours. Furthermore, lymphocytes taken from young mice did not provoke tumours when injected into normal mice. The inescapable conclusion was that a high level of Bcl-2 is not in itself sufficient to cause cancer, even though it significantly raises the odds. For a Bcl-2 over-expressing cell to initiate malignant growth, it must acquire additional mutations.

The researchers found that mutations which activate another oncogene, myc, are particularly potent for converting a cell carrying a bcl-2 mutation cell into a lymphoma. The myc gene promotes cell division. Thus the combination of a growth-promoting mutation (myc) and an apoptosis-inhibiting mutation (bcl-2 ) is a potent recipe for cancer.

Blocking the demolition of the cell

The experiments described above made it clear that defects in apoptosis not only contribute to cancer but also thwart radiation and other anti-cancer therapies. This realisation has galvanised hundreds of researchers around the world to concentrate on clarifying the biochemical pathways that control apoptosis.

Three key players in cell death have been identified in genetic studies, by Dr Robert Horvitz's laboratory in Boston, of a tiny nematode worm called Caenorhabditis elegans . Two "killer genes", ced-3 and ced-4, are essential for the death of superfluous cells in the worm, but their activity can be blocked by the ced-9 gene.

Vaux realised that ced-9 might well be the worm equivalent of bcl-2 and produced dramatic proof of this while working as a postdoctoral fellow in California. He showed that bcl-2 could be used in worms to inhibit cell deaths. This experiment vividly illustrates how strongly the genetic program for apoptosis has been conserved during hundreds of millions of years of evolution.

The killing process is becoming better understood. When researchers in Boston characterised ced-3, they found that it was very similar to mammalian protein-degrading enzymes (proteases) of a new class now called caspases. It is the caspases that dismantle the cell by cleaving many key structural and regulatory proteins.

To prevent their inadvertant suicide, cells manufacture caspases as inactive precursors. It is the conversion of these inert precursors to active caspases that marks the cell for death. In the worm, the ced-4 protein mediates this irreversible step, and the first human ced-4 relative has recently been found. Researchers in the Division are avidly searching for others and trying to determine how Bcl-2 frustrates their activity.

Assisted cell suicide

Apoptosis can have elements of murder, as well as suicide. Certain cells produce a 'death warrant' - a protein that binds to a specific receptor spanning the surface membrane of other cells. The portion of the death receptor that dangles inside the cell then attracts inactive caspases and converts them to an active form. Intriguingly, Strasser has shown that this particular highway to cell death bypasses Bcl-2. Thus, once the caspases have begun the cell's execution, Bcl-2 can no longer provide a reprieve. Tracing the different pathways to cell death and identifying the various check-points are important goals of the Division.

Sibling rivalry in the Bcl-2 family

Over the past few years researchers have come to realise that our cells contain a whole family of Bcl-2-like proteins. Remarkably, while some members also enhance cell survival, others instead favour cell death. Members of the two factions can grapple with each other and block each other's function. This tug of war appears to serve as a cellular thermostat, determining whether the cell will continue to thrive or instead activate the caspases and commit suicide.

Scientists in the Division have recently identified a new pro-survival member of the family called bcl-w, and a new pro-death member, dubbed bim. Why are there multiple genes of each type? The most likely answer is that the different related genes are "tuned' to respond to the special needs of different types of cells.

A powerful way to test this notion is to determine what happens to mice engineered to lack the gene of interest (knock-out mice). For example, mice lacking a functional bcl-2 gene develop normally, but die early in life due to a fragile immune system. Thus, bcl-2 is more important for survival of lymphocytes than other cell types. The Division's researchers are now developing mice lacking either the bcl-w or the bim gene and should soon know where in the body each of these genes is most critical.

Viruses reveal novel inhibitors of apoptosis

Paradoxically, dying is a good defence against viruses! Because these intra-cellular parasites can replicate only in living cells, altruistic suicide of the infected cell precludes infection of neighboring cells. To circumvent this defence, viruses have developed clever strategies to block apoptosis. One, initially identified in viruses of insects, involves a gene known as IAP (inhibitor of apoptosis).

Dr Vaux and his colleagues have discovered that mammalian cells also produce several IAP-like proteins. It is still unclear how IAPs block cell death, but their action appears to be very different from that of the Bcl-2 family. Researchers in the Division are creating IAP knockout mice to explore how they function.

Apoptosis and disease

Defects in the cell death process are implicated in many serious diseases. Genetic accidents that block apoptosis contribute to the development of many cancers, and thwart their treatment, as the Division's seminal work on follicular lymphoma has shown. At the opposite end of the spectrum, neurodegenerative disorders such as Parkinson's disease or motor neuron disease, as well as auto-immune disorders such as diabetes, all involve premature or excessive cell death.

By clarifying the underlying mechanisms of apoptosis, researchers in the Molecular Genetics of Cancer Division hope to pave the way for improving treatment of both cancer and the degenerative diseases. Pinpointing the specific apoptosis pathway blocked in a disease will expose its Achilles heel. In the short term, these insights should help doctors select the optimal conventional therapy; in the longer term they offer the prospect of new modes of treatment and preventative measures.

A new fight begins against breast cancer

Breast cancer is most common cause of death from cancer in women in Australia and the rate of incidence appears to be increasing. The major known risk factors are age and a family history of the disease. Recent research has identified several genes (eg BRCA1 and BRCA2) whose mutation appears to greatly increase the probability of developing breast cancer. However, these mutations may explain as few as 1 to 2% of all breast cancers. Mammography has greatly facilitated early detection of the disease, but there is an urgent need for the development of novel therapies. For this to happen, it is essential to understand more about the normal development of breast tissue and the events which lead to malignancy.

Dr Jane Visvader and Dr Geoffrey Lindeman have recently established within the Molecular Genetics Division a new laboratory dedicated to understanding the molecular basis of breast cancer. They are seeking to identify the 'master' genes which direct epithelial cells to become specialised breast tissue and to understand in intimate detail the controls exerted over the survival and division of breast epithelium. New strains of mice which either over- or under-express candidate regulatory genes in their breast tissue are being created, in the hope of gaining a better understanding of the disease process and providing early markers. Dr Lindeman has a joint appointment with the Department of Clinical Haematology and Medical Oncology of The Royal Melbourne Hospital.

The Visvader/Lindeman laboratory is the latest to join a new 'Institute without walls' created by the State Government - the Victorian Breast Cancer Research Consortium. Over a 10 year period, the charter of the Consortium is to derive fundamental new knowledge that will lead to better treatment and, hopefully, reduced incidence of this major killer. The other laboratories of the Consortium are led by Dr Jane Armes (Peter MacCallum Cancer Institute), Dr Erik Thompson (St Vincent's Institute of Medical Research) and Dr Evan Simpson (Prince Henry's Institute of Medical Research).