Breast Cancer: Knocking Out a Killer

Few bits of DNA have as bad a reputation as BRCA1 and BRCA2, the breast cancer susceptibility genes. Women with inherited alterations in either one are at a high risk of developing breast and/or ovarian cancer, and these mutations are responsible for 30 to 80 percent of all hereditary forms of the diseases. Genetic testing can detect cancer-predisposing mutations, but a positive result gives only an estimated risk. It cannot reveal whenif evercancer might develop.

For this reason, the BRCA genes have become synonymous with Damocles sword, hanging over the lives of women with family histories of breast cancer. The disease burden is impressive: a report from the National Cancer Institute (NCI) estimates that about one in eight women in the U.S. (approximately 13 percent) will develop the disease during her lifetime.

Mutations in the BRCA genes are responsible for only a small fraction of these cases. But for researchers, they represent one of the few handles they can grab at to study the molecular pathways behind breast cancer. Thus, the BRCA genes are among most molecular oncologist's top-10 favorite subjects. A glance at the medical literature reveals that BRCA1 alonethe better studied of the two geneshas been the subject of more than 2,000 papers published in peer-reviewed journals to date.

From this work, scientists have learned that the protein BRCA1 encodes normally helps guard DNA against damage from free radicals, radiation and chemicals. Such damage can jeopardize genetic information and alter the normal function of cancer-related genes, eventually leading to the appearance of tumors. Whereas some cellular machinery actually kills off cells that have accumulated too much damage, the BRCA1 protein seems to act like a plumber, fixing breaks as they occur along the genome.

A study in the May issue of the Proceedings of the National Academy of Sciences confirmed this idea, showing that the BRCA1 protein physically binds to damaged DNA and then recruits other molecules to assemble the equivalent of a DNA-repair kit. Scientists further demonstrated that the absence of the BRCA1 protein does not directly cause cancer but makes cells more susceptible to mutations that in turn can lead to malignant transformations.

Much of this knowledge, though, comes from studies done in test tubes and cells. In the body, cancer is a multistep process: many changes, driven by consecutive genetic alterations, must take place before a dutiful cell can escape the redundant mechanisms in control of its growth; others disruptions are required to foul the immune system's surveillance and lay new blood vessels to feed tumor cells. As a result, scientists desperately need living models to explore the process of cancer development in its full complexity.

Making Living Models

Genetic engineering has allowed scientists to make their own living models for studying a host of diseases. In many cases, they create strains of animals (usually mice) in which they "knock out" a single gene, destroying its function. Also, they can introduce mutations at specific sites to reproduce those behind human diseases. Hundreds of strains of so-called knock-out rodents exist today and represent the ultimate tool for studying cancer.

"In these animals you can study the molecular signaling pathway and the other genes involved in cancer progression," says Jeff Green, a researcher at the National Cancer Institute in Bethesda, Md. "You can observe the effect of different factors, such as hormones, on the tumor growth, and you can cross animals carrying different mutations to see their effect combined."

Given the usefulness of knock-out mice, it would seem natural that researchers would try to breed a strain of mice lacking BRCA1. But when they first tried it in 1996, the results were disheartening. Embryos in which both copies of the gene were knocked out died before birth because a catastrophic number of DNA breaks quickly accumulated. Rodents lacking only one copy of the gene survived but were no more susceptible to tumors than were normal mice.

Three years ago, however, researchers at the National Institute of Diabetes and Digestive and Kidney Diseases at the NIH found a way out of this bind by placing the genetic equivalent of a time bomb in the BRCA1 gene. They buried short DNA strings, called Lox, inside the BRCA1 gene of mice. Like hidden charges, Lox cut and destroy the DNA around them when special, tissue-specific proteins prompt them. The Lox-laden rodents were healthy at birth and developed normally; when the females became adults, however, the Lox bomb detonated in the mammary gland, destroying the BRCA1 gene. Several months later, some of the female mice developed breast tumors similar to those seen in women.

"We found a lot of genetic and chromosomal changes in these tumors that helped us to figure out their origin," says Chu-Xia Deng, senior author of the study, which was published in Nature Genetics. The researchers discovered, for example, that a well-known tumor suppressor gene, called p53, is often mutated together with BRCA1 in mammary tumors. Deng says that other scientists are now interested in these animal models to test anticancer drugs in preclinical studies.

From Transgenic Mice to Treatments

More recently, a serendipitous discovery inspired a different solution for creating live animal models for breast cancer. Research groups at both Columbia University in New York City and at the Dana Farber Cancer Institute in Boston published results this month in Genes & Development. "We took the idea from a paper describing the case of a woman, affected by breast cancer, that had inherited a particular mutation in BRCA1," Argiris Efstratiadis, leader of the Columbia research group, explains.

This mutation had not completely destroyed BRCA1 but resulted in a protein half as long and lacking some of its function. So instead of completely knocking out the BRCA1 gene, the scientists "knocked in" a carbon copy of this mutation to the mouse genome. Such genetically modified animals produced only the half-length protein, developed normallyat least for some strainsand after a period of latency, sometimes developed large breast tumors. What's more, BRCA1 was mutated in all tissues, so these miceunlike the BRCA1 knock-out micealso suffered from other cancers, including sarcomas, lymphomas and carcinomas.

"Although BRCA1 is usually referred as a breast cancer susceptibility gene, it is actually a [general] cancer susceptibility gene," Efstratiadis says. Indeed, several studies have shown that people with an altered BRCA1 have a small increased risk for developing a particular tumor of the peritoneum, or the tissue lining the stomach cavity. So, too, male carriers have a higher-than-normal risk of developing prostate cancer. As a result, these transgenic animals provide a promising model for studying the involvement of BRCA1 in other cancers.

Unfortunately, neither animal model so far mimics the most awful feature of breast cancerits extreme tendency to spread to other tissues, especially the bones. "Among the hundreds of transgenic mice that have been made to study tumors, I haven't yet seen a true good model for bone metastasis," Green says. But these rodents may help in the development of future high-tech methods in cancer management.

Today thumbnail-size silicon chips called microarrays allow researchers to analyze rapidly and simultaneously the activity of thousands of genes in a cell or tissue all at once. Using microarrays, scientists can count which genes are on and off and how much each gene is "working" in a small sample of normal or tumor tissue. Soon these chips will be routine devices in many diagnostic laboratories, offering genetic fingerprints of patients' tumors. With these data, oncologists may well be able to create custom cancer treatments.

For now, scientists will need to learn more more about the intricate molecular pathways of cancerand transgenic mice will be their best allies in the quest.