The cellular events underlying metastasis show certain similarities to those that occur during development - not too surprisingly, since both processes are characterized by cell proliferation.

Increased mobility is one of those similarities. When cells become mobile during embryonic development, they usually go only where they are supposed to. But when cells become mobile during metastasis, it's like a teenager with a driver's license - they are off on the body's highway, the bloodstream, wreaking havoc and causing terror.

Researchers from the University of Texas M.D. Anderson cancer center in Houston have gained new insights into the cellular events that cause increased mobility and underlie cancer metastasis, as well as developmental processes. Those insights were published in the October 2004 issue of Nature Cell Biology under the title "Dual regulation of Snail by GSK-3b -mediated phosphorylation in control of epithelial-mesenchymal transition."

The epithelial-mesynchemal transition, or EMT, is "one important step in metastasis," said Mien-Chie Hung, professor and chair of the department of molecular and cellular oncology at M.D. Anderson and senior author of the study. "Epithelial cells are all together in a sheet and can't move. But when they go to the mesenchymal state, they can move around and get into the bloodstream," because they become less adhesive.

That increased cellular mobility during both development and metastasis is regulated by the same molecular mechanism: the down-regulation of a protein known as E-cadherin. E-cadherin, in turn, is suppressed by the protein "snail."

However, more detailed study of snail had been difficult, since snail is quite the misnomer. Not only does the protein get cells moving in development and metastasis, but, with an average cellular life expectancy of just 25 minutes, snail also lives fast and dies young.

The beginnings of the studies presented in Nature Cell Biology might not seem overly auspicious - the scientists tested 26 cancer cell lines for snail protein and could not find it anywhere.

However, "that wasn't terribly unexpected. Most of the literature says it's difficult to detect," Hung said. The usual way to detect snail is by looking for its mRNA, rather than the protein. "We found it very difficult to detect the protein until we added an inhibitor. Then, it became very easy."

Getting Snail To Sit Still Elucidates Regulation

To gain further insight into snail's function in the EMT process, the researchers needed to make a more stable version. Hung's group managed to produce that version through site-directed mutagenesis, replacing a single amino acid in snail's sequence. The mutation led to a much more stable version, which in turn allowed Hung and his group to probe the protein's cellular fate.

"Snail is a transcriptional factor and supposedly just existed in the nucleus," Hung said. His group showed a more complex story: In the nucleus, snail initially is phosphorylated by GSK-3b (GSK stands for glycogen synthase kinase). The initial phosphorylation induces translocation into the cytoplasm, where snail first is phosphorylated again by GSK-3b and then tagged with ubiquitin to mark it for destruction (that ubiquitin process netted its discoverers the 2004 Nobel Prize in chemistry).

The point mutation of snail protein prevented the chain of action that usually leads to snail's quick disposal at its first step, which also meant that the mutant protein remained in the nucleus instead of being transported to the cytoplasm.

When the researchers studied breast cancer cells expressing the mutated snail, they found that in those cells, snail accumulated in the nucleus. Furthermore, E-cadherin was suppressed and the cells moved around more than their cousins expressing wild-type snail. Hung believes that the findings will not be restricted to breast cancer cells, but turn out to be more generally applicable - a belief supported by previously published research that shows increased levels of snail in several cancer types.

Nevertheless, several findings suggest that it is snail's upstream regulation by GSK-3b, rather than the protein itself, that goes awry in cancer metastasis. For one thing, the researchers found no mutations of snail in cancerous cell lines they studied, suggesting that the increased cellular mobility in cancer is not caused by a defective snail protein. Also, during metastasis, the down-regulation of snail's target E-cadherin usually is transient, as is the increased motility of the cancerous cells.

Those findings spurred Hung and his group to directly test how manipulating those upstream processes would affect snail. They found that they could affect the levels of snail in cell lines by manipulating levels of GSK-3b, suggesting that targeting GSK-3b offers possibilities for manipulating the fast-lived snail.

Hung summarized the work's potential applications: "One may be able to enhance GSK-3b to down-regulate snail and enhance E-cadherin. [E-cadherin upregulation] has been shown to slow tumor formation in an animal model. If you tweak that further upstream by targeting GSK-3b, maybe you have a better chance" of manipulating the pathway.

In the meantime, Hung said his group has been getting "almost daily requests" for the mutant snail, which has opened up the possibility of studying the EMT in greater detail, hopefully with payoffs in both cancer and developmental research. "People had been trying to induce EMT, but it was almost impossible to do. There was no good model, and now there is."