Alex Robinson is a freelance medical writer working in Ottawa.
Canadian Medical Association Journal 1995; 153: 1123-1124
[résumé]
Leder, he says, was one of the first researchers to introduce human oncogenes into the mammary tissue of mice. These transgenic mice were able to pass on the foreign gene to their progeny. The aim of the research was to develop a model that could explain how oncogenes affect mammary epithelial cells at the molecular level.
"One of the genes we focused on," says Muller, "is called neu in mice and c-erbB-2 or HER-2 in humans; it's known by a variety of names but it's essentially the same gene." The gene drew attention because it is amplified and therefore overexpresses its protein in about 30% of cases of sporadic (i.e., noninherited) breast cancer. More than 80% of breast cancer cases are sporadic. The neu gene codes for a receptor that binds with certain growth factors and stimulates cells to divide. "To sum up a large body of literature, the higher the level of expression of this gene the poorer the prognosis."
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Early experiments
Muller's early work involved putting a mutated version of neu into the genetic material of healthy mice. Offspring carried the mutated gene, which coded for a constitutively active form of its protein that continually signals mammary epithelial cells to divide. The promoter that was used to make the mutated neu gene produce its protein -- the mouse mammary-tumour-virus promoter -- caused the mutated gene to express high levels of the protein. The proto-oncogenic version of neu is to the mutated form, Muller explains, as a car with an idling engine is to one in which the accelerator is pushed to the floor. Although it is not clear whether the human equivalent of neu is mutated in human breast cancer, a mutated version of the gene was used initially to make the function of the gene more apparent.
"The results of these studies were really quite dramatic," says Muller. "Almost every mammary epithelial cell became transformed, which is unusual in a transgenic-mouse model." Here was direct evidence that the neu gene was "a very potent transformer of mammary epithelial cells." Moreover, these findings correlated nicely with the results of studies on the overexpression of neu in human breast tumours.
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Refining the model
Although the mutated version of the neu gene, so effective at transforming mammary epithelial cells in mice, does not appear in human cells, Muller explains, the wild-type neu protein is overexpressed in human breast cancer. For the past 5 years Muller and his colleagues at McMaster University have been developing "a comparable mouse model using just the wild-type neu protein - the `normal' protein, we call it."
Muller has received funds from several sources for his work. He has been awarded a grant of $53 000 over 2 years from the Cancer Research Society of Canada, $89 000 over 3 years from the Medical Research Council of Canada, $120 000 over 3 years from the Breast Cancer Research Initiative and $117 000 (US) over 4 years from the US Department of Defence. If this seems a long list, the work is expensive: "We spend $100 000 a year on mice alone," Muller says.
The return on this investment includes new insight into how breast cancer starts, improved understanding of the prognosis of the disease and, potentially, new avenues of treatment.
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Genesis
Muller found that fewer mammary tumours developed in mice that overexpressed the wild-type neu protein than in those that carried the mutated neu gene. Longer studies, however, showed that animals carrying the wild-type gene were prone to a higher incidence of metastasis. Muller notes that the latter finding is consistent with the clinical literature on human breast cancer, which shows that a high expression of neu is associated with a poor prognosis. However, the finding that fewer tumours occurred in mice with the wild-type neu gene made Muller wonder whether other factors might have a hand in triggering tumour growth. He notes that a recent study has yielded "very exciting results" that suggest that an inframe deletion of part of the neu protein DNA sequence that occurs just outside the transmembrane domain may be the cause of about 60% of mammary tumours arising in the mice that overexpress the neu protein.
An inframe deletion, Muller explains, is a deletion that affects the regulation of a protein but not its essential function. The transmembrane domain is the segment of a protein that anchors it to the cell membrane. "This area of the molecule hasn't been inspected for mutation in human breast cancer," says Muller. "So it is conceivable that comparable mutations occur in humans in that region. We're very interested in this possibility and are looking into it right now."
Moreover, he adds, it is possible that such a mutation could be passed from one generation to the next. Support for this idea comes from the discovery of a similar mutation in a molecule implicated in the inherited form of endocrine neoplasia type 2A.
The inframe deletion found in the neu protein occurs in an area of neu that is conserved in three closely related members of its gene family. Those other members, the receptors erbB-1, erbB-2 and erb-4, are also overexpressed in human breast cancer. This suggests that these genes may also be involved in the genesis of breast cancer and points the way for further investigation.
Meanwhile, another series of experiments has shown that neu is involved in the c-src pathway. Muller explains that c-src was one of the first oncogenes ever cloned or described and that it is overexpressed in about 50% of human breast tumours. "We think an interaction between these two genes is often very critical in producing cell proliferation. So we are looking into this, too."
The mouse models Muller and his colleagues have developed allow them to enhance or suppress the expression of oncoproteins. Making one gene overexpress its protein while disabling another can stall the appearance of breast cancer in mice, sometimes indefinitely. But in crossbred mice that overexpress the products of both neu and myc -- the genes are from two different pathways -- tumour genesis is accelerated. The myc gene is a transcription factor that is also overexpressed in human breast cancer.
Genes Muller is studying in this way include the p53 tumour-suppressor gene and c-src. "These are clinical correlates. What we'd like to do is understand how the genes interact and lead to cancer."
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Diagnosis and treatment
The finding of an overexpression of the neu protein in a woman with breast cancer has diagnostic significance: "The prognosis would be less favourable," Muller says.
But there is hope that further research will help to turn this around. Muller speaks of studies that show that the immune system recognizes and kills tumour cells that have been made to express cytokines, nonantibody proteins that modulate the immune response. "The bottom line is that the mouse model can be used to test the efficacy of various novel therapies for breast cancer. This includes antibody therapy, which is being studied in clinical trials in the United States."