Oct. 17, 2000 U-M researchers prove ability of RhoC GTPase to trigger inflammatory form of disease
ANN ARBOR, Mich. - University of Michigan researchers have found that a gene recently implicated in liver, skin and pancreas cancer can cause an especially deadly kind of breast cancer, and may help explain why it grows, spreads and sometimes proves deadly so quickly.
The finding, published in the Oct. 15 issue of the journal Cancer Research, confirms the team's earlier suspicions that a gene called RhoC GTPase is a key factor in inflammatory breast cancer. Just over a year ago, they published a study showing that the gene was over-expressed in 90 percent of tumor samples from women with the aggressive disease, several times more often as in women with non-inflammatory forms of the disease.
Now, the authors say, they have shown that too much RhoC GTPase production in otherwise normal cells causes the kind of rapid establishment of cell colonies, invasive tendencies and ability to move that characterize inflammatory breast cancer, or IBC.
They also found that, implanted into healthy mice, normal cells with extra RhoC GTPase activity can prompt breast tumors to form - though not as often as implanted tumor cells. This suggests that the gene, while key to the disease's progress, has some undetermined partners.
"This is the first time that the RhoC gene has been implicated in breast cancer, and we suspect that its importance may go beyond the inflammatory form of the disease to include other aggressive breast tumors," says author Sofia Merajver, Ph.D., M.D. associate professor of internal medicine in the U-M Health System and director of the Breast and Ovarian Risk Evaluation Clinic at the U-M Comprehensive Cancer Center.
Adds Kenneth van Golen, Ph.D., co-lead author with Zhi-Fen Wu, M.D., "This discovery raises the possibility of a future test or therapeutic agent that could help physicians and patients launch a counterattack as aggressive as the disease itself." Such tests or treatments are probably years off, he added, but their development relies on evidence like that in the new study.
Inflammatory breast cancer accounts for 6 percent of all breast cancer diagnoses in the U.S. each year, but its speed and ability to metastasize make it much harder to treat. Only 45 percent of women with the disease are alive and disease free after five years and optimal treatment.
IBC is known as the most deadly form of locally advanced breast cancer. The disease's name comes from the red color and other changes it produces in breast skin, including nodules, puckering and nipple retraction. By the time it's diagnosed, IBC has almost always spread to the lymph nodes and often to other parts of the body - suggesting its cells quickly develop the ability to leave the primary tumor, travel through the body and grow blood vessels elsewhere.
Despite its deadly nature, little has been known about IBC's genetic underpinnings. The U-M team has probed that mystery for several years, most recently with studies of which genes were over-active in the tumors of women treated for various forms of the disease at the U-M Comprehensive Cancer.
They found that the RhoC GTPase gene was over-expressed - or transcribed extra times to produce surplus RhoC GTPase protein - in 90 percent of IBC tumors. This molecular-level difference seemed to help explain why IBC cells and non-IBC cells don't look different under a microscope, but behave very differently in the body.
RhoC GTPase protein is known to help cells form and arrange the "skeletal" protein actin, which helps form the infrastructure for cells that are dividing to make new cells, extending themselves in a particular direction, attaching to a surface, and stimulating new blood supply routes. An increased ability to perform all those activities is a hallmark of cells that have been transformed into cancer cells - they tend to reproduce without brakes and form colonies, move around the body, cling to each other and to anchor locations, and form blood vessels to feed themselves.
Armed with that knowledge, the U-M team set out to study the influence of the over-expressed RhoC gene by itself by inserting the gene from tumor cells into normal breast cells, in a process called transfection. They then compared those cells' behavior with that of normal cells, as well as cells transfected with a control gene, and cells originally grown from IBC tumor tissue. The transfected cells produced about as much of the RhoC GTPase protein as IBC cells.
They found that extra RhoC alone was enough to cause the transfected cells to form more colonies of new cells than non-transfected cells, from 6 to 176 times as many colonies depending on how much of the protein and how many copies of the gene were present. Even the lowest colonization rate was almost as high as that of tumor cells.
More crucial to understanding the problem of IBC, the extra RhoC gave the cells a much greater ability to move, or grow across a barrier, than normal cells - about as much invasive ability as tumor cells. The RhoC-transfected cells were also much more likely to move large distances across a surface than the other transfected cells. The source of that movement, concentrated areas of actin called focal adhesion points and stress fibers, was clearly visible in images of the RhoC cells and tumor line cells, but not the others.
Finally, the team tested the RhoC cells' ability to start tumors in the breast areas of female mice, compared with tumor cell lines. A quarter of the mice that got the RhoC cells formed tumors, while none of those implanted with normal breast cells did.
"Overall, our result suggests that there are other key genetic factors to be found," explains Merajver. The underexpression of a tumor suppressor gene called LIBC, also found by the U-M team in the prior study, may be one. Already, the team is continuing its study of all the factors.
The study was funded by the National Institutes of Health, the Susan G. Komen Breast Cancer Foundation, and the U.S. Army Breast Cancer Research Program.
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