Almost from the moment cytologists first noticed that cancer cells contain an abnormal number of chromosomes, researchers have debated the significance of the observation. It's a classic "chicken/egg" dilemma, and one that has aroused passions on both sides of the issue. Does chromosomal instability cause cancer, or is the condition of aneuploidy merely a consequence of predisposing gene mutations? A flurry of recent research is beginning to clarify the issue and in many cases is placing aneuploidy in the front and center of the carcinogenic process.
Within the past few months, several research teams have shown direct links between cancer syndromes and abnormalities in the surveillance system that ensures accurate chromosome segregation during cell division. Nazeen Rahman, Ph.D., of the Institute of Cancer Research in Sutton, England, and her colleagues reported in the November 2004 issue of Nature Genetics that inherited mutations in a key protein involved in ensuring accurate chromosome segregation during mitosis can lead to childhood cancers.
The researchers studied eight families that had at least one family member affected by mosaic variegated aneuploidy (MVA), a recessive condition in which cells in many tissues exhibit varying degrees of aneuploidy. They found mutations in the gene BUB1B in five of the eight families, including in two children who had developed a rare cancer, rhabdomyosarcoma.
In contrast, no mutations in the gene were seen in 200 patients with sporadic cancers and 384 normal control subjects studied. BUBR1, the protein product of BUB1B, is known from animal studies to delay chromosome segregation until all chromosomes are attached to the spindle apparatus that pulls paired chromosomes to opposite poles in dividing cells. Mutations in the gene lead to abnormal chromosome segregation, with daughter cells receiving too few or too many chromosomes.
"This study provides the most direct evidence to date linking aneuploidy to cancer development," said Hongtao Yu, Ph.D., assistant professor of pharmacology at the University of Texas South-western Medical Center in Dallas.
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But MVA is rare and Rahman's sample size was small. Despite years of looking, few mutations in spindle checkpoint proteins had been found in sporadic cancers, causing many to doubt their importance for the majority of cancer cases. Even in experimental systems, linking chromosomal instability to cancer has been difficult. But Yu, who studies regulation of the mitotic spindle, said that the reason it has been difficult to link defects in chromosome segregation and cancer is that most mutations that cause chromosomal instability also kill the cell.
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"These [spindle checkpoint] genes are very much essential for basic aspects of cell biology," he said. "If you mutate these genes to cause too much of a loss of function, you probably cause gross chromosomal missegregation, which would probably compromise cell viability. You probably need specific mutations of these genes in combination to give you cell viability and also probably subtle missegregation that gives you aneuploidy in the long run. Those are the mutations that contribute to cancer progression. It is actually very difficult to design experiments to show that unequivocally. It is easier [to study the mutations] that nature creates over time."
By taking a closer look at protein interactions at the mitotic spindle, Yu and others have now found subtle yet direct links between defects in spindle checkpoint control and inherited, as well as more common, sporadic cancers.
Within a single month, two groups reported links between the breast cancer predisposing-genes BRCA1 and BRCA2 and the mitotic spindle apparatus, leading investigators to speculate that both play a role in ensuring proper chromosome segregation. In the December 7 issue of Proceedings of the National Academy of Sciences, Yu and his collaborators Rui-Hong Wang, Ph.D., and Chu-Xia Deng, Ph.D., of the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, Md., reported that mice deficient in the full-length BRCA1 protein had decreased levels of proteins involved in the spindle checkpoint, and they further showed that BRCA1 directly regulates transcription of those genes. Mouse breast cells deficient in BRCA1 failed to properly segregate chromosomes and activated p53-induced apoptosis in laboratory studies. The researchers suggested that chromosome instability in BRCA1-deficient cells could, over time, lead to mutations in oncogenes, such as p53.
"I think, in time, mutations in BRCA1 will create an opportunity for chromosome changes, for other mutations to happen, and if one of those changes happens to be in p53 or perhaps other oncogenes, the cells overcome the apoptotic pathway and have a chance to grow," Deng said.
The result could explain the long latency in BRCA1 tumor formation and the propensity for BRCA1-associated tumors to acquire mutations in p53 as well, he said. More than 90% of BRCA1-associated tumors in humans also have mutations in p53, whereas only about 40% of sporadic cancers do. He also points out that if mice with p53 mutations are crossed with BRCA1-deficient mice, the resulting double mutants all get tumors and do so much faster than animals with single mutations.
Similarly, Ashok Venkitaraman, Ph.D., and his colleagues the Medical Research Council Cancer Unit in Cambridge, England, used small interfering RNA targeted to BRCA2 to show that loss of the BRCA2 protein impairs the completion of cell division in mouse embryo fibroblasts. The researchers suggest in their October 29 article in Science that BRCA2 may have a role in regulating cytokinesis because the protein localizes to the spindle apparatus.
Together, the two studies link chromosomal abnormalities in two hereditary breast cancer syndromes to chromosomal instability and help bolster the idea that aneuploidy helps drive the oncogenic process.
Further support comes from the laboratories of Christoph Lengauer, Ph.D., Bert Vogelstein, M.D., and their colleagues at Johns Hopkins University in Baltimore. The groups selected a subset of 100 of the more than 1,000 genes that can cause chromosomal instability in animal model systems. The researchers sequenced those 100 genes in 24 aneuploid colorectal cancer cell lines and identified 19 acquired mutations in five genes representing three pathways to chromosome instability.
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"There is probably not one gene or a few genes that can cause chromosomal instability, but probably many, many different ones and all of them in small portions," said Lengauer. "We believe that chromosomal instability is a driving force, that it is necessary to have, along with other mutations that are required to develop a tumor."
The same research team identified mutations in the human CDC4 gene that cause chromosomal instability in some cancer cells and are present in precancerous adenoma lesions, but they could not explain how the mutations in the gene lead to instability. But in the December 9 issue of Nature, Allan Balmain, Ph.D., and his colleagues at the University of California at San Francisco presented evidence that hCDC4 (also known as Fbxw7) is a p53-dependent tumor suppressor that, when lost through mutation, leads to chromosome instability by inducing a protein regulator of the spindle apparatus. The researchers showed that mice with mutations in hCDC4 and p53 develop a different spectrum of cancers than mice with p53 mutations alone. They argue, in agreement with Lengauer and his colleagues, that loss of hCDC4 could be a driving force in tumorigenesis.
"We have now shown that in about 40% of colorectal cancers that instability actually causes mutations in certain genes," said Lengauer. "What we think is you get a mutation in one of those genes that causes chromosomal instability and that drives the process of tumorigenesis and allows other mutations to happen that are required to get a tumor.
"Experiments which we have done indicate that chromosomal instability is extremely early," he added. "It is potentially possible that instability comes first, but that is very difficult to prove."
However, Carlos Cordon-Cardo, M.D., Ph.D., of Memorial Sloan-Kettering Cancer Center in New York, and Scott Lowe, Ph.D., of Cold Spring Harbor Laboratory in New York, argue that their experiments show that, at least in some cancers, chromosomal instability can be explained as a consequence of disruption of the retinoblastoma gene. In an August 12 Nature paper, the scientists demonstrated that the mitotic checkpoint protein Mad2 is a direct target of E2F, which in turn is disregulated in cells with retinoblastoma defects. The researchers created retinoblastoma pathway defects in normal and transformed cells that led to elevated levels of Mad2 and subsequent aneuploidy.
Although Yu, who has shown that it is a decrease in Mad2 expression that causes aneuploidy, has no explanation for their findings, he agrees with Lowe and his colleagues that in sporadic cancers aneuploidy is probably not the first step in tumor formation.
"I still think aneuploidy is a relatively late event in cancer formation," Yu said. "The other hit probably happens earlier, and aneuploidy is the second hit. It is a way to speed up chromosomal instability. Vogelstein's group has shown in colon cancer [that chromosomal instability] appears relatively early, but it's probably not the first hit. I think it facilitates tumor formation; it gives the extra speed to accumulate mutations that then lead to cancer."
Regardless of whether chromosomal instability happens early or late, researchers seem to agree that aneuploidy must now be recognized as a major force in oncogenesis, with possible implications for cancer treatment.
A tantalizing suggestion of the possibility of driving aneuploid cells into apoptosis was suggested by Don Cleveland, Ph.D., of the University of California at San Diego. In the June 8 issue of the Proceedings of the National Academy of Sciences, Cleveland and his colleagues reported that using small interfering RNAs to reduce levels of the checkpoint proteins BubR1 or Mad2 in human cancer cells or inhibiting BubR1 kinase activity provoked apoptosis. He suggests that manipulating the checkpoint proteins in aneuploid tumors has potential to treat certain cancers.
"I think it could work," Lengauer agreed.
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