Researchers from Massachusetts General Hospital (MGH) have identified a new tumor suppressor gene that appears to be inactivated in gliomas, a deadly form of brain tumor. Levels of the protein coded by the gene, called ING4, appear to correspond with tumor aggressiveness, with the lowest levels seen in the highly malignant glioblastomas. In their report in the March 18 issue of Nature, the researchers also describe how ING4 appears to suppress the growth of new blood vessels to feed the tumor, a process called angiogenesis.
ING (for INhibitor of tumor Growth) is a family of proteins known to be essential to the activity of the powerful tumor suppressor p53. The research team, led by Igor Garkavtsev, MD, PhD, of the Steele Laboratory in the MGH Department of Radiation Oncology, searched a gene database to identify genes with a similar structure to the previously identified ING1.
"The ING4 gene had been cloned simultaneously in both laboratories at the MGH and at the National Institutes of Health," says Garkavtsev. "Its functional role in the prevention of tumor progression had not been investigated. When we found that malignant gliomas have strikingly lower levels of ING4 and knowing that ING4 regulates angiogenesis, this sparked interest toward potential targets that were independent of p53."
The research team first tested samples from 50 tumors of various malignancy grades for evidence of ING4 gene expression. They found that gene expression was two to three times lower than normal in lower-grade tumors and up to six times lower in the high-grade glioblastomas. Similarly another test that compared tissue from brain tumors with samples of nearby normal tissue found abundant ING4 levels in the normal tissue but significantly less in the tumors.
To investigate the protein's effect on angiogenesis, the researchers created lines of tumor cells that were manipulated to express levels of ING4 higher and lower than found in control tumors. Cells from each of these lines and from unaltered tumors were implanted into cranial windows, transparent compartments placed on the brains of mice to allow observation of blood vessel growth.
Implants of cells with suppressed ING4 expression grew much faster and developed more complex blood vessel networks than did the controls. The implants that overexpressed ING4 grew much more slowly and showed significantly less blood vessel growth. Further examination with an advanced imaging technology confirmed that suppression of ING4 was associated with more mature and complex vascular networks, while overexpression kept vessels from significant development.
Another set of experiments indicated that ING4 acts to suppress the activity of a protein called NF-kappa B, which is known to stimulate genes that are overactive in many forms of cancer. Loss of the ING4 suppressor causes overproduction of the NF-kappa-B-responsive gene for interleukin 8 (Il-8), which is known to stimulate angiogenesis. Knowing the mechanism by which gliomas are able to support their growth offers some possible drug targets – including restoration of ING4 function, inactivation of Il-8 production through NF-kappa B, or blocking the Il-8 sensitivity of tumor cells.
Additional authors of the Nature paper are senior author Rakesh Jain, PhD, director of the Steele Laboratory; Sergey Kozin, DSc; Lei Xu, MD, PhD; Frank Winkler, MD, PhD; and Edward Brown, PhD, all of the Steele Lab; and Olga Chernova, PhD, and Gene Barnett, MD, of the Cleveland Clinic. The research was supported by grants from the National Cancer Institute.
Massachusetts General Hospital, established in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of more than $400 million and major research centers in AIDS, cardiovascular research, cancer, cutaneous biology, medical imaging, neurodegenerative disorders, transplantation biology and photomedicine. In 1994, MGH and Brigham and Women's Hospital joined to form Partners HealthCare System, an integrated health care delivery system comprising the two academic medical centers, specialty and community hospitals, a network of physician groups, and nonacute and home health services.
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