Reactivating A Critical Gene Lost In Kidney Cancer Reduces Tumor Growth

In the August 15 issue of Clinical Cancer Research, investigators report that suppression of this gene, which makes secreted Frizzled-Related Protein-1 (sFRP-1), appears to be one defining epigenetic event in the development and progression of clear cell renal carcinoma, which is responsible for at least 80 percent of kidney cancers. The researchers also found that sFRP-1 controlled 13 tumor-promoting genes along the powerful "Wnt" signaling pathway, whose activity has been linked to a number of different cancers, of which colon cancer is best known.

"For all intents and purposes, sFRP-1 acts as a key tumor suppressor gene in clear cell renal cell carcinoma — it puts a brake on Wnt, stopping dangerous oncogenic signaling to the cancer cell, thus blocking tumor growth and metastasis," explained the study's senior investigator, John A. Copland, Ph.D., a molecular biologist at Mayo Clinic Cancer Center. Michelle Gumz, Ph.D., a former postdoctoral researcher at Mayo Clinic, is the lead author of the study. She is now at the University of Florida.

The findings may also be relevant to other cancers, such as breast, ovarian, prostate, bladder, lung and colon cancers, in which loss of sFRP-1 function is common, Dr. Copland explained. "Through understanding the important role sFRP-1 plays, we may be able to eventually tailor human therapies to restore its function in this type of kidney cancer and in other cancers," he says.

Kidney cancer incidence and related deaths have been increasing by about 2 percent per year for the past six decades, for reasons that are not fully understood but may be related to smoking and other environmental factors. More than 50,000 new cases of the cancer are expected in 2007, with almost 13,000 deaths. Kidney cancer tumors are often found when they are advanced and extremely difficult to treat: two-year survival at that stage is only 12 percent whereas kidney cancer that has not yet spread has a five-year survival of up to 75 percent.

To aid diagnosis and treatment, Dr. Copland and a team of researchers have been developing a panel of biomolecular markers for kidney cancer. They are comparing tissue from renal cell tumors with normal kidney tissue from the same patient, searching for "master" genes that control signaling pathways that are involved in cancer development and progression. A master gene, if altered, would shut down the cascade of cancer-causing genes that it controls. "There are likely to be a number of molecular events that lead to cancer development, so we want to find as many genes as we can that we might shut down, like Wnt, or turn back on, like sFRP1," Dr. Copland says.

To find out which genes are activated and which are silent, in normal versus cancerous kidney tissues, the research team used three independent sets of patient kidney tissue samples from Mayo Clinic College of Medicine, in Jacksonville, and from The University of Texas M. D. Anderson Cancer Center in Houston. In one sample, they found that gene expression of sFRP-1 was "down-regulated," or turned down low or off, in 15 of 15 patients. They also found that activity of the gene decreased as much as 70 times the level seen in normal tissue.

In a second set of 33 patient samples, the researchers looked for evidence of messenger RNA (mRNA) activity; mRNA, transcribed from sFRP-1, is the blueprint that cells need to produce sFRP-1 proteins. Every stage of kidney cancer showed decreases in sFRP-1 mRNA, some as much as 140-fold.

Investigators then searched for differences between sFRP-1 protein levels in normal versus cancerous cells within another set of 39 matched patient samples. They found that 70 percent of the cancerous samples showed a total loss of sFRP-1 proteins, and the other 30 percent had very little of the vital protein in cells. "The take home message is that there was almost a complete loss of sFRP-1 protein in most of the renal cell carcinoma samples we studied," Dr. Copland says. "If no protein is being produced, the Wnt signaling pathway is free to activate other molecules that can cause cancer."

Finally, researchers used a common experimental technique to see what would happen if they reactivated sFRP-1 in tumor cells. They had discovered that, in clear cell renal carcinoma, sFRP-1 is silenced through a process known as methylation, blocking the ability of transcription factors and proteins from binding on and activating genes, thus "silencing" them.

Restoring sFRP-1 protein expression by a technique of transfecting the sFRP1 gene into human renal cancer cells was remarkably effective, Dr. Copland says. Growth of tumors decreased by at least 90 percent in sFRP1 transfected patient-derived cancer cells in the laboratory, he said, and Wnt regulated oncogenes, such as c-myc, were suppressed compared to untreated cells.

In mice, tumors in which sFRP-1 function had been restored for seven weeks were an average of 3 percent the size of tumors in untreated animals and many tumors disappeared completely. "This was a dramatic reduction," Dr. Gumz says. "Our report is the first to show anti-tumor activity of sFRP-1 in an animal model and to clearly implicate Wnt as an oncogenic signaling pathway in renal cell carcinoma."

Understanding and targeting the Wnt signaling pathway with drugs has been a goal of researchers worldwide, Dr. Copland says. "There are at least 20 known Wnt molecules and five members of the sFRP family, and we are slowly beginning to understand how they work together." Anti-methylation agents are now being tested in various clinical trials, he added, saying, "Given results of this study, that kind of strategy might be useful. There are few effective therapeutic options for metastatic clear cell renal cell carcinoma that result in increased longevity and quality of life. Restoration of sFRP-1 function represents a possible therapeutic target."

The findings might also be helpful in identifying which type of kidney cancer a patient has developed. The researchers found that loss of sFRP-1 is common in clear cell renal cell carcinoma as well as in the next most common form of renal cell carcinoma, the papillary subtype. But loss of this gene is not seen in chromophobe renal cell carcinoma and benign oncocytoma. "Although biopsies are not performed for renal cell carcinoma currently, creating molecular signatures unique to the subtype of renal cell carcinoma using genes such as sFRP1 and removing a small piece of tumor tissue by biopsy could save some patients from surgical removal of a benign mass," Dr. Copland says.

The study was funded by National Cancer Institute grants, the James and Esther King Biomedical Program of the Florida Department of Health, and the Dr. Ellis and Dona Brunton Rare Cancer Research Fund. Co-authors include, from Mayo Clinic College of Medicine, Jacksonville: Pamela A. Kreinest, Dr. April C. Childs, Leandra S. Belmonte, Shauna N. LeGrand, Dr. Kevin J. Wu, Dr. Alexander S. Parker; from Mayo Clinic College of Medicine, Rochester, Minn.: Dr. Hongzhi Zou and Dr. David A. Ahlquist; Dr. Christopher G. Wood, from the University of Texas M. D. Anderson Cancer Center, Houston, Texas; Dr. Bruce A. Luxon and Mala Sinha, from the University of Texas Medical Branch; Galveston, Texas; and Dr. L-Z Sun, from the University of Texas Health Science Center, San Antonio, Texas.