[Editor's Note: This is the third article in a three-part series on cancer research in Connecticut. The first article, on chemotherapies and diagnostics, appeared in Volume 9, No. 2; the second, on epidemiologicol studies, in Volume 10, No. 3.]
NOT so long ago, researchers dreamed of finding a gene responsible for human cancer. Since then, many exciting discoveries have been made in cancer research. We now know that cancer has a genetic basis, and researchers have identified genes responsible for several types of this deadly disease. These advances in our understanding of how normal cells are transformed into cancer cells has been made possible by the application of molecular genetics to the study of cancer biology, a field called molecular oncology.
Despite these great strides in understanding how cancer develops, clinicians cannot yet effectively treat most forms of cancer. The challenge today is to use the information provided by the field of molecular oncology to develop more effective therapeutic agents. Research in molecular oncology is producing exciting results in several Connecticut institutions: examples of the work currently under way at two of them are described below.
Anti-cancer drugs that are currently available work by preventing proliferation of the tumor cells, but they act non-specifically and therefore also affect proliferating non-tumor cells, causing severe side effects in patients. The next generation of drugs will inhibit specific biochemical processes that occur only in cancer cells. Because they will affect only tumor cells, these mechanism-based, anti-cancer agents will be more potent and less toxic to the recipient than conventional drugs.
Bayer Pharmaceutical Division's program to develop mechanism-based, anti-cancer drugs is built on strong extramural collaborations, especially with Onyx, a small biotechnology company located in California. Scientists at Onyx discover new targets for therapeutic intervention within cancer cells, and scientists at Bayer develop the compounds intended to act upon those targets.
Researchers at Onyx and Bayer are developing therapies for solid tumors, such as colon, pancreatic, and lung cancer. The first line of therapy in treating solid tumors is to surgically remove the tumor and then treat the patient with chemotherapeutic agents to kill any residual tumor cells. In many instances, not all of the tumor cells are killed, and many solid tumors reappear within five years. It often is not possible to repeat the same chemotherapy treatment, because the drugs are so toxic or because the cells become resistant to their effects. In addition, tumors often metastasize, or spread, to other locations, causing cancer in other internal organs, especially the brain, bones, or lungs.
The goal of the Bayer group is to develop a drug that extends life and improves the quality of life for patients with metastatic tumors after they are treated with chemotherapy. Since the drug would have to be administered to patients over a period of years, its safety must be clearly demonstrated; in addition, it is preferable that the drug be one which can be taken orally. The drug also must affect a target that is selective for the tumor and is essential for tumor progression.
Control of cell division is the key to controlling the growth of tumors. Normal cell growth is controlled by genes, in particular oncogenes and tumor suppressor genes. In the past 20 years, approximately 70 oncogenes have been identified; more recently, researchers have also identified about a dozen tumor suppressor genes. The results of this work suggest that both types of genes influence the development of cancer. As a series of genetic defects accumulate over time, some turn on, or activate, oncogenes while others inactivate tumor suppressor genes, resulting in uncontrolled proliferation of the cells and, eventually, tumor formation.
Cells receive signals from hormones, growth factors, other chemicals, and physical contact with other cells, which tell the cell whether to remain quiescent or divide. The process of transmitting those signals to the nucleus, where the decision is made whether to divide or not, is called signal transduction. This process is controlled by the products of oncogenes and tumor suppressor genes. If the signal transduction process goes awry, because oncogenes are continuously activated or tumor suppressor genes are defective, the cell divides without control. Correcting the faulty signal transduction process should stop the uncontrolled division of the tumor cells.
According to Gunther Karmen, Section Head for Cancer Research at Bayer's West Haven laboratory, the first step in finding a drug to interfere with the defective signal transduction process is to identify a molecule in the process that is the product of a mutated gene. "One of the targets we have chosen is ras, an oncogene that is mutated in 30% of all cancers-50% of colon, 90% of pancreas, and 50% of lung cancer," says Dr. Karmen.
The ras gene has been studied for many years; it was cloned 12 years ago and its function in the signal transduction process was identified 10 years ago. Recent research shows that mutations in ras are relevant to the development of tumors, and scientists at Bayer confirm that ras is a target for therapeutic intervention in cancer. "When we implanted a ras-mutated cell line into mice, the mice developed tumors, and when ras was repaired, the cells no longer formed tumors," says Dr. Karmen. "We decided ras would be a good target."
Once the target molecule has been identified and its function in the signal transduction process has been confirmed, the next step is to identify a compound that will affect the activity of the target. "We investigate all possible sources of drugs. We screen libraries of existing drugs and natural products, and design new drugs based on the structure and function of the target," says Dr. Karmen. "We try to find a highly potent compound that will selectively modify the biochemical activity of the target."
There is a service unit within Bayer for high-volume screening of potential products. The Bayer group picks several compounds that seem to be good candidates as therapeutic agents against the target molecule, then tests them in cell culture to see if they are effective at killing human tumor cells or cells into which has been inserted the oncogene that codes for the target. They also test compounds in animal models of cancer, with an emphasis on metastasis models, since the goal is to treat metastatic cancer.
Scientists choose the most promising product for preclinical testing, which includes extensive cell culture and animal testing to determine how the compound acts once it is in the body, and to uncover any possible side effects. After many years of experiments, testing, and review by the federal Food and Drug Administration (FDA), the drug is eligible for testing in a small number of patients. If the results are promising, further clinical trials are conducted, followed by further review by the FDA. Drugs targeting oncogenes, such as those being developed at Bayer to target the ras oncogene, are in the early stages of development and will require several years of study before they are available to patients.
Tumor suppressor genes are also under investigation as targets for drugs to treat cancer. Research in this area is relatively new, so much remains to be learned about how these genes might be used in cancer therapy. Tian Xu, an Associate Professor of Genetics working in the Boyer Center for Molecular Medicine at the Yale University School of Medicine, is trying to identify tumor suppressor genes and then elucidate the functions of those genes during normal development of the organism.
"Our goal is to identify genes responsible for cancer in humans," says Professor Xu, "but because we cannot experiment with human beings, we work with experimental animals." Professor Xu's choice of an experimental animal, Drosophila, or the common fruit fly, may seem surprising, and, in fact, met with some skepticism at first. "Fruit flies do not get tumors," he was told. "Why would you want to work in that system?" His response was that no one had found tumors in Drosophila because no one was looking for them. "It seems obvious now," he says. "Tumor suppressor genes regulate proliferation of cells. During development there is a continuous need to signal cells to stop proliferating and start differentiating, so there must be genes like tumor suppressors filling that role." Professor Xu's first goal was to identify those genes in Drosophila.
Professor Xu identified a gene called lats, which, when mutated, forms benign tumors in all tissues in Drosophila. He cloned lats and found that it codes for an enzyme involved in signal transduction. His next questions were: Does a form of lats occur in humans? And does this gene have any relevance for human tumors?
Professor Xu's laboratory screened libraries of human and murine (rodent) genes and found genes with sequences similar to that of lats in both humans and mice. To test whether the functions of these genes were similar to that of lats, he produced Drosophila that were mutated for lats and introduced the human gene into them. "Inserting the human lats-like gene into the mutated flies rescued the animals," says Professor Xu. "The proliferation defect and all developmental defects were fixed." These experiments proved that a form of the lats gene does exist in humans.
Professor Xu's laboratory is now testing to see if lats is relevant for human tumors by screening human tumors to look for mutations in lats. They are also systematically dissecting the biochemical pathway of this tumor suppressor gene to see if the pathway is conserved between species.
Michael Reiss, an Associate Professor of Medicine in the Department of Internal Medicine (Section of Medical Oncology) at the Yale University School of Medicine, has helped to solve a paradox that had puzzled researchers for years.
He is studying a chemical called transforming growth factor ß (TGFß), which is produced by tumor cells and affects the surrounding tissue, helping the tumor invade and metastasize. TGFß also inhibits cell division. "We wondered, how can these cells produce TGFß, exist in high concentrations of this growth inhibitor, and still multiply out of control?" says Professor Reiss.
As his group discovered, the answer lies in defective TGFß receptors. Most cancer cell lines (cancer cells that are grown generation after generation in the laboratory) either have defective TGFß receptors or lack them completely, and the genes encoding those receptors are mutated. "Without a functional receptor, the cancer cells do not recognize the TGFß, and can be continuously bathed in it without any effect," explains Professor Reiss.
Primary tumor cells-cells taken directly from patients-also have defective TGFß receptors. Twenty percent of the esophogeal cancers that Professor Reiss's group have investigated have lost the receptor entirely, and five out of the fifteen breast cancers that they have studied have mutated receptors. They are now investigating at which stage of tumorigenesis this loss of receptor expression occurs. "We are also investigating whether loss of TGFß receptor expression can be used prognostically, that is, to tell if a tumor is going to be particularly aggressive, so we could deal with it at an early stage," states Professor Reiss. The goal is to introduce a normal copy of the TGFß receptor gene into tumor cells, to make the cells responsive to TGFß, and, ultimately, to bring cell division under control.
David Stern is an Associate Professor of Pathology and Biology at the Yale School of Medicine. One of his areas of research is a growth factor receptor called erbB2, which is found in abnormally high amounts on cancer cells. The gene for erbB2 is amplified 100 times in 25% of breast and ovarian cancers. "This is not a case of a mutated gene making a defective protein," says Professor Stern. "The gene is normal and so is the protein, as far as we can tell. There is just too much of it."
The results of cell culture experiments indicate that having too much of the erbB2 receptor causes cells to overproliferate and form tumors. Professor Stern's laboratory is working to understand the biology of this receptor with respect to breast cancer, both to understand the disease and to help develop new therapies. "There is a lot of evidence that this gene is involved in cancer," explains Professor Stern. "By understanding the system, we hope to be able to predict how the patient will do and how she will respond to drugs."
Many advances in cancer research have been the result of work with tumor viruses. Oncogenes first caught the attention of researchers because tumor viruses encode them; only later were oncogenes found to be important in the development of human cancer. The first tumor suppressor gene, p53, was discovered because its protein product bound to a protein made by a tumor virus.
Daniel DiMaio, Professor of Genetics at the Yale University School of Medicine, is investigating the relationship between a certain class of viruses, called papilloma viruses, and cancer. There is a strong epidemiological link in humans between papilloma virus and cervical cancer; in fact, the cell abnormalities that show up in a PAP smear are caused by papilloma virus infection.
"We are studying an animal virus that works somewhat differently from the known human papilloma viruses, but could provide important information as to how viruses affect cell growth," Professor DiMaio says. "Viral proteins can turn on cell growth; studying how they do that can teach us what happens in cancers to cause uncontrolled cell growth. In addition, some viral proteins can turn off cell growth, and by studying that process, we hope to determine mechanistically how to treat cancer."
The DiMaio laboratory has identified a papilloma virus protein that turns on a cellular receptor that normally senses the presence of growth stimulatory molecules in the environment. According to Professor DiMaio's research, the viral E5 protein binds to the receptor and tricks it into thinking the cell is flooded with these growth stimulatory factors. As a consequence, the receptor delivers a sustained signal to the cell that results in uncontrolled proliferation and tumor formation. Quite the opposite occurs when another papilloma virus protein, the E2 protein, is expressed in some human cancer cells. Professor DiMaio has shown that the E2 protein activates an intracellular tumor suppressor pathway that tells the cells to cease proliferation. It may someday be possible to develop drugs or other treatments that block the interaction of E5 with its receptor or mimic the effect of E2. Such treatments may inhibit cancer cell growth. "Our experiments have provided new information about the mechanisms by which viral proteins affect cell growth," says Professor DiMaio. "These studies have also provided new insights into the cellular pathways that control cell growth. Manipulation of these pathways will ultimately form the basis for rational treatment of many kinds of tumors, including some that are unrelated to viral infection."
The past 20 years of research on the functions of oncogenes combined with more recent work with tumor suppressor genes have greatly increased our understanding of how cancer develops. Much remains to be explored in the field of molecular oncology and the next several years will bring more exciting developments, including, researchers hope, the promise of effective new therapies with which to treat cancer.--Lisa Christenson, freelance science writer.
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