Dean A. Haycock

Special to BioWorld Today

Although cancer's status as the No. 2 killer might indicate otherwise, the process by which a healthy cell turns into a cancer cell is not a simple one. Cells undergo multiple changes during their transformation from the cellular equivalent of Dr. Jeckyll into Mr. Hyde. Unfortunately, unlike Robert Louis Stevenson's characters, transformed cells do not revert to their original, nonmalignant state as readily as Hyde turned back into Jeckyll.

Understanding what factors contribute to the aggressiveness of cancer cells and the tricks they use to sustain themselves could, naturally, lead to better cancer therapeutics. A promising lead in ongoing efforts to identify key factors controlling the development of aggressive cancers is the progression elevated gene-3 (PEG-3), identified in 1997 by Paul Fisher and his colleagues. (See BioWorld Today, Sept. 2, 1997.)

"One of the questions that has been around for a long time is, 'What are the genes that control the conversion of a normal cell to a benign cancer cell to a highly aggressive cancer cell?'" Fisher, a professor of clinical pathology at Columbia University's College of Physicians and Surgeons in New York, told BioWorld Today.

He and his associates used a method they developed called subtraction hybridization to identify genes that are differentially expressed under different physiological conditions. "It is a strategy in which we first subtract away genetic information to enrich for differentially expressed genes and then we actually display this information using a technique that is quite commonly used - differential display." This technique, RSDD, provides a "snapshot" of genes that are altered as a consequence of changes in properties of cells. In this context, the researchers were specifically looking for genes that made a rodent cancer cell more aggressive.

They exploited the fact that the rat cancer cells they studied underwent a stable genetic change. These cancer cells maintained their aggressive phenotype no matter how long they were maintained outside of an animal and they recapitulated the same aggressive phenotype when put back into an animal. This enabled the researchers to identify PEG-3, which was shown to directly correlate with cancer aggressiveness.

"As a cancer became more capable of forming tumors and developing more rapidly, this gene was expressed at elevated levels," Fisher said. "But when we first did the study we didn't know whether the gene was associated with phenotype or causative."

Fisher and his collaborators move this story along with publication in the Dec. 21, 1999, issue of the Proceedings of the National Academy of Sciences of an article titled, "PEG-3, a nontransforming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis."

This paper departs from the previous one by addressing the fundamental issue of what happens when you overexpress the PEG-3 gene in a normal or a cancer cell. "This gene, or a homologue of this gene, will be expressed as cancers become more aggressive," Fisher said. "Also, the gene can selectively turn on that cascade in a target cell that is already a tumor but it can't turn it on in a cell that is a normal cell."

Classic oncogenes can turn normal cells into cancer cells and make them more aggressive. "But," Fisher noted, "PEG-3 is a gene that apparently will do nothing to a normal cell, but when put into a cell that already has genetically programmed tumorigenic potential, it exacerbates that genotype and makes it much worse." This finding applied to both in vitro and in vivo experiments. "Not only did it work in rodent cancers," Fisher recalled, "but if you put it into a human cancer, you develop the same aggressive phenotype."

The researchers showed that forcing tumorigenic rodent cells and human cancer cells to express PEG-3 decreased the time it took these cells to form tumors in nude mice and produced larger tumors that contained more blood vessels. They also showed it was possible to block these PEG-3-related effects using an antisense expression vector that prevented the expression of the PEG-3 gene in progressed rodent cancer cells. There was, furthermore, a direct correlation of cancer aggressiveness in cells expressing PEG-3 with RNA transcription, messenger RNA levels and secretion of vascular endothelial growth factor (VEGF).

Angiogenesis, or neo-vascularization, of course, is an absolute requirement for the growth of tumors beyond a cubic millimeter or so. VEGF stimulates endothelial cells to form new components of blood vessels.

Fisher et al. made use of the fact that normal cells can't grow when they are suspended in a matrix such as agar, whereas cancer cells can. "We found that when we expressed PEG-3 in the cancer cells, their colony-forming ability in agar increased," Fisher explained. "We know that forming more colonies in agar has nothing to do with angiogenesis. This suggests that PEG-3 may work in two areas to accentuate cancer. One is to switch on genes that makes cancer more aggressive. The other is to modify the blood supply to the cancer. If you put both of those hits together in the same cancer cell, you have potent ways of making a cancer more aggressive."

But how can a single genetic element like PEG-3 change a cancer cell into a more aggressive cancer cell? Fisher suspects PEG-3's ability to promote cancer aggressiveness might be related to its ability to turn on other genes not related to VEGF as well as to its ability to turn on VEGF, which allows even greater elaboration of the cancer phenotype.

Fisher added that this approach "can be used to identify genes and products for directly inhibiting angiogenesis or actually inducing it. You can actually look in both directions, which is exciting."

The group is now looking for other genes that might be involved in fine-tuning the aggressive cancer phenotype. Fisher hopes to "define either small molecules or antisense agents, or whatever novel technologies are out there, to inhibit this gene, its homologues and downstream genes and thereby inhibit cancers from progressing."

The National Institutes of Health, the Chernow Endowment and the Sam Waxman Cancer Foundation helped fund the research.