SAN FRANCISCO, CA — A tiny protein called RhoC found in breast tumors may someday give doctors and patients an early warning system that could spot dangerously aggressive breast cancer long before it begins to spread, and identify the need for aggressive treatment.
A test to detect the protein is still more than a year away from clinical trials. But promising early results show that RhoC can serve as a marker for breast tumors that are most likely to spread, or metastasize — even identifying them when they’re less than a centimeter in diameter.
Physicians from the University of Michigan Comprehensive Cancer Center developed the test based on their prior research on the RhoC gene, and proved its effectiveness in 182 tissue samples from the U-M’s breast cancer library. Results will be presented April 9 at the meeting of the American Association for Cancer Research.
The test detected invasive cancer that had the potential to metastasize with 88 percent specificity, and had 92 percent specificity for tiny tumors that had already metastasized. Samples of normal breast, benign breast cysts, or non-invasive breast cancer had little RhoC.
“This is a very promising marker for small but invasive breast cancers that may metastasize, which right now are hard to identify,” says lead author Celina Kleer, M.D., an assistant professor of pathology at the U-M Medical School who specializes in breast cancer. “While more research is needed before clinical testing can begin, we hope it will help identify early-stage cancer that could be vulnerable to aggressive treatment, perhaps with drugs that target Rho protein.”
Kleer and her colleagues, including experienced RhoC researchers Sofia Merajver, M.D., Ph.D., associate professor of internal medicine, and Kenneth van Golen, Ph.D., assistant professor, embarked on the study to find out how much RhoC was produced in different kinds of breast cancer cells, compared with normal breast cells.
Previously, they had shown that the RhoC gene was overexpressed in inflammatory breast cancer, a particularly deadly variety that grows and metastasizes quickly. Overexpression of the gene, they believed, might also occur in other kinds of aggressive breast cancer – leading to larger quantities of the RhoC protein in cells of those cancers.
RhoC, whose full name is RhoC-GTPase, is an enzyme involved in changing the internal skeleton of a cell — changes that allow a cell to polarize or move. That ability is important in muscle cells, which produce a lot of RhoC. But in cancerous non-muscle cells, RhoC is key to the structural changes that give a cell the ability to break off from a tumor, float through the body in the bloodstream, and take hold in a satellite location – in other words, to metastasize.
In finding the inflammatory breast cancer correlation, the U-M team was the first to show that RhoC, already implicated in liver, pancreas and skin cancer, was also involved in breast cancer. U-M researchers, led by Merajver, then showed that transplanting the RhoC gene into normal breast cells in mice transforms those cells into cancerous ones with metastatic potential.
The new research started with the development of a RhoC test. With help from the U-M Protein Structure Facility, and their knowledge of the RhoC gene, the team created an antibody that would latch on to RhoC protein anywhere in a tissue sample. A stain specific to the antibody then allowed the U-M researchers to see how concentrated the protein was in different tissue samples from breast tumors and surrounding areas.
The breast cancer library at the Cancer Center provided 182 tissue samples from 164 patients whose breasts had been biopsied at the U-M, as well as information about whether they had cancer or benign breast disease like fibrocystic changes. Samples of breast cancer tissue were accompanied by information describing exactly what variety of cancer it was, how large the tumor was, whether it was invasive (spreading beyond the layer of cells where it started and into healthy tissue), what stage it was in, and whether it had metastasized through the body.
The cancerous samples included everything from ductal carcinoma in situ (a low-grade localized condition), to stage IV invasive ductal carcinomas that had spread to lymph nodes or other parts of the body. The wide spectrum allowed the team to look for differences between cancers of different sizes, stages, types and levels of invasiveness.
Antibody staining revealed the expected high levels of RhoC in muscle and blood vessel cells surrounding the breast tissue, and none in normal breast tissue. But the concentrations of the reddish-brown stain in the cancerous tissue varied greatly. When Kleer correlated the samples that stained darkest with their clinical characteristics, she discovered a connection.
“We found RhoC only in invasive cancers, and it almost always correlated with the presence of metastases. Very few non-metastatic cancers contained high levels of RhoC,” she says. “The level of RhoC expression also increased as the stage of the breast cancer increased, which is another confirmation that it’s a marker of more aggressive cancer. We had enough samples from invasive metastatic cancers of less than one centimeter in size to show that RhoC is highly specific for those tumors, but we’d like to look at more samples to be sure.”
Kleer, Merajver, van Golen and their colleagues are preparing to examine even more breast samples for the presence of RhoC, to see if their initial results hold up. The team is also in the process of planning clinical studies on the predictive power of RhoC.
The study was funded by the National Institutes of Health, the Department of Defense’s breast cancer research program, and a grant from the John and Suzanne Munn Endowment at the U-M Comprehensive Cancer Center. In addition to Kleer, Merajver, and van Golen, the study’s authors are Zhi-Fen Wu, M.D.; Yanhong Zhang, Ph.D.; and Mark Rubin, M.D.
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