Breast Cancer: The Big Picture Emerges

Courtesy of Cold Spring Harbor Laboratory  IDENTIFYING NEW CANCER GENES: (1) Clinicians biopsy cancerous (left) and normal (right) tissues from patient. (2) DNA microarrays containing thousands of individual human genes are exposed to a mixture of labeled dna samples. (3) Red spots indicate genes amplified in cancer cells, green spots show genes deleted. (4) Both classes of genes become potential targets for new diagnostic or therapeutic anti-cancer strategies. In 1994, discovery of the

Feb 10, 2003
Ricki Lewis
Courtesy of Cold Spring Harbor Laboratory
 IDENTIFYING NEW CANCER GENES: (1) Clinicians biopsy cancerous (left) and normal (right) tissues from patient. (2) DNA microarrays containing thousands of individual human genes are exposed to a mixture of labeled dna samples. (3) Red spots indicate genes amplified in cancer cells, green spots show genes deleted. (4) Both classes of genes become potential targets for new diagnostic or therapeutic anti-cancer strategies.

In 1994, discovery of the breast cancer 1 (BRCA1) gene fueled media reports of a single cause behind a single disease. Since then, a far more complex story has emerged, with evidence for both single genes contributing great effects, and many genes with small but additive effects. Many molecular roads, scientists now know, lead to breast cancer.

Only 5% of breast cancers are the consequence of germline mutations in a single gene. More commonly, cancers arise as somatic mutations, and many of these are probably polygenic in origin. "The risk of cancer is probably influenced by many unknown genes, with individual effects that are much smaller than the impact of BRCA1 or BRCA2; their collective contribution is large," says Colin Begg, the head of epidemiology and biostatistics at Memorial Sloan-Kettering Cancer Center in New York.

"SHOW ME THE PROTEINS" The new view of breast cancer genesis has its roots in basic cell biology. "BRCA1 and BRCA2 are exceptions, found by identifying families with many cases and using positional cloning to get to the genes," explains Alan D'Andrea, a pediatric oncologist at the Dana-Farber Cancer Center in Boston. "A new way to find cancer genes is to see which signal transduction pathways they are part of, then show which genes increase cancer risk." And pathways mean proteins. "Proteomics is the way to go at this in a big way. 'Show me the proteins,'" he says.

Proteins implicated in breast cancer include enzymes, growth factors, receptors, cell cycle regulators, and adhesion molecules. Two major classes of genes orchestrate their control. "Tumors arise when the normal pathways that regulate cell growth and cell death are disrupted," says pathology professor Jonathan R. Pollack, Stanford University School of Medicine. "Mutations can make oncogenes overly active, or tumor suppressor genes less active." Oncogenes may be amplified or overexpressed, tumor suppressor genes deleted or underexpressed, sometimes silenced with methyl groups. "In general, these are all independent mechanisms that alter gene expression," Pollack adds. Mutations contribute sequentially to the growth and spread of cancer, in a cascade of altered function.

There are more breast cancer genes than anyone thought, says D'Andrea. Like other cancer genes, those that lie behind breast cancer may sabotage the cell cycle, response to growth signals, DNA repair or apoptosis, while bestowing powers of angiogenesis, tissue invasion, and metastasis. Likely suspects include the genes that encode cyclins, kinases, caspases, and cadherins.

But other candidate genes may be more specific, encoding proteins that help remodel the breast from a fatty pad to a milk machine. During late pregnancy, cells of mammary epithelial buds divide to form ducts, ignoring apoptosis signals and infiltrating surrounding stromal tissue, building a local blood supply, not unlike cancer, although breast cells specialize for their short-term job. For example, integrins and laminins control how the epithelium interacts with the extracellular matrix, and insulin-like growth factor-I and a hedgehog receptor oversee crosstalk between the epithelium and the surrounding stroma. Cancer arises in rare stem cells nestled in the duct epithelium, and may originate from deranged signaling.

SINGLE GENES, LARGE EFFECTS Researchers discover breast cancer genes in several ways. Two sources: families with several cases of early-onset breast cancer, but who have normal BRCA1 and BRCA2 genes; and other disorders that confer greatly increased risk of breast cancer. In both cases, the culprits may be genes whose products interact with either BRCA1 or BRCA2 protein, which are part of the DNA repair machinery.

Consider ATM and CHEK2, proteins that function at the crossroads in the cell cycle, when the cell "decides" to either repair DNA or die.1 Normally, ATM phosphorylates CHEK2, which in turn phosphorylates BRCA1. Mutation of the CHEK2 gene causes 2% of breast cancers in Scandinavia, less elsewhere. Mutation of the ATM gene causes ataxia telangiectasia; its symptoms include immune deficiency, chromosome breakage, and increased sensitivity to ionizing radiation, in addition to increased breast cancer risk. Similarly, Fanconi anemia would likely cause breast cancer if patients lived long enough, D'Andrea discovered.2 Five Fanconi anemia genes encode proteins that form a cluster that activates a sixth protein, which in turn activates another protein that binds BRCA2.

Like the BRCA genes, the retinoblastoma 1 (RB1) gene encodes a tumor suppressor protein vulnerable to disruption by other proteins. Sometimes, the RB1 gene is normal, but a gene that activates the RB1 protein, RB1CC1, is deleted.3 "Loss of RB1CC1 correlates with aggressive cell proliferation. Introduction of RB1CC1 suppresses the cell proliferation of some cancer cell lines," says Tokuhiro Chano, associate professor of clinical laboratory medicine at Shiga University of Medical Sciences in Tokyo.

Systematic searching among chromosome regions commonly deleted in breast cancer cells is another approach to finding tumor suppressor genes. For example, Michael Wigler and colleagues at Cold Spring Harbor Laboratory compared the genomes of breast cancer and normal cells, and zeroed in on part of chromosome 8.4 They identified the DBC2 (deleted in breast cancer 2) gene. "We probed the region for candidate genes, tested each one, and determined the frequency with which each is involved in cancer, is mutated, or deleted. None of the other genes were mutated," Wigler explains, making a long story very short.

The genetic glitches behind some cases of breast cancer are so novel that they evade studies of families, lost chromosome segments, and even measures of gene expression. This is the case for a protein called p27. An oncogene product called Akt kinase phosphorylates p27, which prevents it from entering the nucleus where it would normally switch off cell division.5 "This scenario occurs in breast cancers that contain excessive Akt activity," says Joan Massague, a Howard Hughes Medical Institute investigator at Sloan-Kettering. "Because all cells share the elements that mediate nuclear import, banishing p27 from the nucleus may operate in other cancers, such as prostate cancer, melanoma, and glioblastoma."

Courtesy of Alan D'Andrea
 THE FANCONI ANEMIA/BRCA PATHWAY is a rare autosomal recessive cancer susceptibility disorder. Eight Fanconi anemia (FA) complementation groups have been defined, and seven FA proteins have been cloned (A, C, D2, E, F, G, and BRCA2). Recent studies indicate that these FA proteins interact in a common signalling pathway. Five of the FA proteins (A, C, E, F, G) assemble in a constitutive nuclear protein complex that is required for the activation (monoubiquitination) of the downstream D2 protein. Activation of D2 occurs after DNA damage or during the S phase of the cell cycle. Monoubiquitinated D2 is targeted into subnuclear foci containing BRCA1, BRCA2, and RAD51. Disruption of this pathway results in the common clinical and cellular abnormalities observed in FA.

MANY GENES, SMALLER EFFECTS For years, evidence for a polygenic origin of breast cancer has been indirect--the clustering of cases in families. DNA microarrays are adding precision by identifying contributory genes. Rene Bernards, a professor of molecular carcinogenesis at the Netherlands Cancer Institute, and colleagues have developed a microarray that probes expression of 70 "signature" genes.6

Using tumor samples frozen more than five years ago, the researchers correlated cDNA gene expression profiles with future metastasis. "We compared patients with extremely good or bad outcomes, and the computer selected 70 out of 25,000 genes, half known, half unknown, each of which had a significant ability to predict outcome," says Bernards. The correlations are sufficiently robust for immediate clinical use of the 70-gene microarray in Europe, but US approval awaits prognostic validation, which is ongoing with colleagues at Massachusetts General Hospital, Bernards says.

A key finding of the microarray study is that the decision to spread is already set in a small tumor. "The classical model is that a tumor starts out without the ability to metastasize to a secondary site, and only if it grows bigger does it progress to metastasis. But we can see if small tumors will form metastases," Bernards says.

The 70-gene microarray correctly predicted outcome in 83% of the cases. With success like that, prognostic profiling with microarrays may ultimately replace traditional indicators, says Martin Widschwendter, a professor of obstetrics and gynecology at University Hospital, Innsbruck, Austria. "Clinicians evaluate histology, grade of differentiation, tumor size, and hormone receptor status," he adds. Widschwendter, along with Peter Jones and Peter Laird at the Norris Cancer Center in Los Angeles, are developing profiles based on cancer- specific DNA methylation patterns.

Nearly a decade after women began asking physicians to test for the breast cancer gene, multigene portraits of the disease are nearing clinical reality. Sums up Wigler, "There will probably be molecular profiling of cancer, rather than one single gene that will be most important. We should consider as much information as possible."

Ricki Lewis ( is a contributing editor.

1. L.C. Brody, "CHEKs and balances: accounting for breast cancer," Nat Genet, 31:3-4, 2002.

2. A.D. D'Andrea, M. Grompe, "The Fanconi anaemia/BRCA pathway," Nat Rev Cancer, 3:23-6, January 2003.

3. T. Chano et al., "Truncating mutations of RB1CC1 in human breast cancers," Nat Genet, 31:285-8, 2002. 4. M. Hamaguchi et al., "DBC2, a candidate for a tumor suppressor gene involved in breast cancer," Proc Natl Acad Sci, 99:13647-52, Oct. 15, 2002.

5. S. Blain, J. Massague, "Breast cancer banishes p27 from the nucleus," Nat Med, 8:1076-8, October 2002.

6. M.J. van de Vijver et al., "A gene-expression signature as a predictor of survival in breast cancer," N Engl J Med, 37:1999-2009, Dec. 19, 2002.