Gene knockouts reveal FoxOs' vital functions in cancer defense, health of stem cells

January 25, 2007

BOSTON--In an elegant, multiple-gene knockout experiment, a team of Boston scientists has discovered that a trio of molecules, called FoxOs, are fundamentally critical in preventing some cancers, maintaining blood vessel stability, and in keeping blood-forming stem cells healthy.

The discoveries reveal potential new targets for cancer drugs and could further research on stem-cell based therapies for degenerative diseases, said the researchers at Dana-Farber Cancer Institute and Brigham and Women's Hospital, who are jointly publishing two reports in the Jan. 26 issue of Cell.

The researchers at Brigham and Women's found that mice engineered to lack genes for the FoxO1, FoxO3, and FoxO4 molecules had serious blood abnormalities. Without the FoxO gene-regulating molecules, the rodents' blood stem cells - master cells that give birth to working blood cells while also renewing themselves ¬- divided too fast and "burned out," said Gary Gilliland, MD, PhD, who is senior co-author of the two papers with Ronald DePinho, MD, of Dana-Farber.

"If we didn't have these FoxO proteins to keep stem cells healthy, it is likely that we wouldn't be able to live for more than a few months," said Gilliland. Lead author of the stem cell paper is Zuzana Tothova, an MD-PhD student at Harvard Medical School working in Gilliland's lab.

In the companion paper, lead author Ji-Hye Paik, PhD, of Dana-Farber and colleagues from the DePinho lab report that the three FoxO molecules, known as transcription factors, normally function as tumor suppressors that override maverick cells threatening to grow too fast and form tumors. When FoxOs are eliminated, it may allow cancer to develop. The mice lacking FoxO proteins developed two types of cancer - thymic lymphoma and hemangiomas, tumors caused by the uncontrolled growth of endothelial cells that form blood vessels.

DePinho's group identified two genes regulated by the FoxO molecules that might serve as points of attack for new cancer drugs.

"This is going to expand our opportunities for drug discovery in what, arguably, is the most important pathway in cancer," said DePinho.

The FoxO1, O3, and O4 transcription factors regulate genes in the complicated cell signaling network known as PI3K-AKT, or simply PI3K. Scientists have discovered that PI3K signaling is intimately involved in fundamental cell processes such as metabolism, aging, and protecting the body against cancer. The PI3K circuit has been found to be disrupted in many forms of cancer, making it a hot topic in cancer research labs and drug company boardrooms.

Based on previous work in his laboratory, DePinho, working with Diego Castrillon, MD, PhD, (who is now at the University of Texas Southwest Medical Center), determined that the three FoxOs had redundant, overlapping functions: To uncover those functions, it would be necessary to engineer mice that lacked all three FoxO transcription factors.

To make the task even more difficult, mice lacking FoxO1 die in the womb. DePinho and Castrillon had to engineer mice whose FoxO genes would function normally during development, but would contain a mechanism allowing them to be switched off in adulthood at the scientists' will. It took DePinho's team about two years to get the system to work, which Gilliland hailed as a "true tour-de-force of mouse genetics."

Mutant FoxOs have been implicated in leukemia, and for Gilliland, who studies blood cancers, the triple-knockout mice were an opportunity to dig deeper into the issue.

Unexpectedly, however, deletion of FoxO1, 03, and 04 caused blood cell abnormalities but not outright leukemia. A bigger surprise was that the blood stem cells "were really in trouble without those transcription factors," he said, dividing too rapidly, losing their ability to renew themselves, and dying out. "This means that FoxOs contribute to the longevity of stem cells, and if you take them away, you dramatically shorten stem cells' lives."

Looking further, Gilliland and his colleagues found that the damage was being caused by reactive oxygen species, or ROS, a toxic byproduct of cells' energy production. When the mice were treated with anti-oxidants, the stem cells regained health and longevity. "So, the FoxOs are acting as natural antioxidants," said Gilliland. Conceivably, he added, drugs could be developed to manipulate the FoxO pathway and extend the lives of stem cells beyond their natural limits, which could aid their use in repairing diseased body tissues.

The results raised an important question: If the PI3K pathway and the FoxO factors are so prominent in cancer, why did the knockout mice lacking FoxO tumor suppressors not develop more types of cancers? It turns out that the cancer-causing pathway operates differently in different types of body tissues. For example, blood vessel cells in the mice's livers became malignant, but the same cells in the lung did not.

"There is a remarkable context-specific aspect to this pathway," DePinho said. "It is wired and regulated differently the same types of cells residing in different types of tissues." This is important knowledge, he said, for further research and for testing novel drugs in the right types of cancers.

DePinho and Gilliland emphasized the importance of the collaboration between Brigham and Women's and Dana-Farber, both of which are affiliated with Harvard Medical School, in producing the results reported in Cell.
-end-
The research was funded by grants from the National Institutes of Health, the Ellison Medical Foundation, and the Leukemia and Lymphoma Society. Gilliland is an Investigator of the Howard Hughes Medical Institute and DePinho is an American Cancer Society Research Professor.

Brigham and Women's Hospital is a 747-bed nonprofit teaching affiliate of Harvard Medical School and a founding member of Partners HealthCare System, an integrated health care delivery network. BWH is committed to excellence in patient care with expertise in virtually every specialty of medicine and surgery. The BWH medical preeminence dates back to 1832 and today that rich history in clinical care is coupled with its national leadership in quality improvement and patient safety initiatives, dedication to educating and training health care professionals, and strength in biomedical research. With $370M in funding and more than 500 research scientists, BWH is an acclaimed leader in clinical, basic and epidemiological investigation - including the landmark Nurses Health Study, Physicians Health Studies, and the Women's Health Initiative. For more information about BWH, please visit: www.brighamandwomens.org.

Dana-Farber Cancer Institute (www.dana-farber.org) is a principal teaching affiliate of the Harvard Medical School and is among the leading cancer research and care centers in the United States. It is a founding member of the Dana-Farber/Harvard Cancer Center (DF/HCC), designated a comprehensive cancer center by the National Cancer Institute.

Dana-Farber Cancer Institute

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