||The role of two closely related proteins, long
suspected of being major contributors to the development of a number of
cancers, has at last been identified. Researchers with the U.S. Department
of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) have
shown that the proteins known as "Ski" and "Sno" block
the "downstream" events initiated by TGF-ß (transforming growth
factor-beta), an extracellular protein responsible for controlling the
growth and differentiation of certain types of cells.
The TGF-ß protein sends a signal that stops the growth of epithelial cells – the cells that line the skin, kidney, glands, lungs, gastrointestinal tract, bladder, and blood vessels. If something goes awry, causing the TGF-ß signal to be blocked, cell growth continues unchecked, giving rise to cancerous tumors. Nearly 90-percent of all human cancers involve epithelial cells.
A research team led by Kunxin Luo, a cell biologist with Berkeley Lab's Life Sciences Division, and an assistant adjunct professor with the University of California at Berkeley, has been able to demonstrate that Ski and Sno proteins interact with a family of proteins called "Smad" to completely shut down the TGF-ß signal. Smad proteins are known tumor suppressors.
"We've shown that Ski and Sno, two oncogene products, directly interact with the protein products of tumor suppressor genes at a common point," says Luo. "This indicates that oncoproteins and tumor suppressor proteins do not act independently. Rather, they regulate one another's function. Our work also suggests that cancer development is a delicate balancing act in which the balance gets tipped the wrong way."
Although the way in which Ski interacts with Smad proteins to control TGF-ß signals continues to be a mystery, Luo and her colleagues have reported that the mechanism behind the effects of Sno is a negative feedback loop (see Science, October 22, 1999).
TGF-ß proteins cannot enter cells and must therefore transmit their signals by attaching themselves to receptor proteins on a cell's outer surface. The signal generated by this interaction is then carried across the cell membrane and into the nucleus via Smad proteins.
Luo and her colleagues discovered that a normal level of Sno inside the nucleus blunts TGF-ß signals, but as the number of Smad proteins increases, the level of Sno drops until it is low enough for the signals to take effect.
"After two hours, the TGF-ß signals have resulted in a marked increase in the expression of the Sno gene," says Luo. "The level of Sno rises until it blocks the functions of Smad. This allows a cell to resume normal growth activity."
When Luo and her colleagues deliberately kept levels of Ski and Sno artificially high, the TGF-ß signal was blocked and cell growth could no longer be restrained. It is unclear at this time to what degree Ski and Sno act independently or in conjunction with one another, and what factors cause the balancing act between these two proteins and the Smad proteins to be tipped in a cancerous direction.
"Cancer is not a simple disease and there are many different pathways through which it can develop," says Luo. "However, knowing the relevant levels of Ski and Sno in a cell gives us a possible tool for early cancer diagnosis."
Luo and her colleagues were able to identify the roles of Ski and Sno in cancer development by working with liver cancer cells of a type known as Hep3B cells. They were looking to identify proteins in the cell nucleus that interacted with the Smad proteins which earlier work had identified as carriers of the TGF-ß signal. To do this, they engineered a Smad protein to host a special molecular "tag" that would be recognized by an antibody. Tagged Smad proteins could then be introduced into cells and subsequently harvested using the antibody. Caught up in the harvest would be any other type of protein that might be attached to the tagged Smads.
"It was an emotional roller coaster ride of anticipation to see what we would find when we analyzed our results," says Luo. "We were quite surprised and very excited when we learned that we had captured oncoproteins."
Specifically in their tests, Luo and her colleagues found Ski and SnoN, the most predominant form of the Sno protein. In the immediate future, Luo plans to investigate the ways in which oncoproteins interact with tumor suppressors to regulate cell growth. This work has implications beyond carcinogenesis.
For example, TGF-ß signals stop the growth of epithelial cells but promote the growth of fibroblast cells. Says Luo, "The discovery of Ski and Sno as blockers of the TGF-ß signal may lead to their applications in the treatment of fibrosis diseases."
TGF-ß signals also promote the differentiation of other types of cells, especially muscle cells. Studies have shown that Ski and Sno affect this process as well – an excess of either results in an abundance of muscle, a deficit yields the reverse. Other important functions of TGF-ß signals include the healing of wounds and the repair of damaged tissue.
Members of the Luo group that worked on the Ski and SnoN oncoproteins were Shannon Stroschein and Wei Wang. Their collaborators were Sharleen Zhou and Qian Zhou from UC Berkeley. Also participating in the Ski experiments were Dan Chen and Eric Martens from UC Berkeley.
Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.