Investigators Use Guilt-By-Association Strategy To Track Potential Cancer Causing Genes

A "guilt-by-association" strategy turns out be just as effective in cancer biology as it is in law enforcement, since cancer-causing proteins that rob cells of their ability to divide normally also depend on co-conspirators. Such molecular partners in crime, once identified, may yield a wealth of information about how a cancer develops and how it best can be treated.

Drs. Harry Hwang and Bruce E. Clurman, researchers at the Fred Hutchinson Cancer Research Center, and colleagues successfully used this approach to identify cancer genes that collaborate with a protein called p27, which is found in abnormally low levels in lymphomas and breast and other cancers.

The team identified 21 potential cancer genes, and as many as 14 of these genes may have not beenpreviously implicated inthe development of cancer. The achievement-made possible by the recent completion of the mouse and human genomes-represents a key step toward the quest by cancer biologists to characterize the cellular pathways that distinguish one type of cancer from another.

The study appears in the current online issue of the Proceedings of the National Academy of Sciences. A copy of the paper is available at http://www.pnas.org. Co-authors include Dr. Matthew Fero also at Fred Hutchinson, and Dr. Anton Berns and his colleagues at the Netherlands Cancer Institute in Amsterdam.

"We know development of cancer requires multiple steps," Hwang said. "Based on work in some human tumors, it appears that the loss of p27 is one significant event. We asked the question, what are the other players in the pathway that lead to tumor formation when p27 is involved?"

According to Hwang, the identification of the complete network of genes required for tumor formation is a first critical step toward developing highly specific anti-cancer therapies.

"We focused on the p27 pathway because the protein has been implicated in the development of many cancers," Clurman said. "In normal cells, the p27 protein blocks cells division in response to many signals and p27 functions as a tumor suppressor. That is, the loss of p27 function is associated with the development of cancer. But how p27 works to suppress cancer is unknown."

Because normal cell division and its perturbation in cancer are complex processes, the team reasoned that much could be learned from identifying proteins in the cell that collaborate with p27 in tumor development.

To identify potential new cancer genes, the team took advantage of a virus known as the Moloney Leukemia Virus, which causes lymphomas in mice. Upon infection, the virus inserts its own DNA randomly into the DNA of the cell. As a result, host cell genes adjacent to the site of viral insertion become activated. In rare cases, the gene near the viral insertion may be what is known as a proto-oncogene, a gene that can promote tumor formation when activated. The advantage of using this technique is that the integrated virus serves as a molecular tag that facilitates subsequent isolation and characterization of the genes adjacent to the viral insertion.

The researchers infected groups of both normal mice and mice deficient in p27 with the virus, then used genome sequence information to determine the identities of the mouse genes adjacent to sites of viral insertion. Although tumors form in both groups of mice, the process is greatly accelerated in the p27-deficient animals.

Since the virus inserts randomly in the mouse genome, the researchers expected to find multiple potential cancer genes whose activation led to lymphoma in both groups of animals. Of most interest were insertion sites common to more than one p27-deficient mouse. With tens of thousands of genes in the mouse genome, insertions in a common site in multiple p27-deficient animals (with few or no insertions in the same region in normal mice) are likely to be near genes that play the most critical roles in p27-mediated cancer development.

"Insertion sites common to two animals are like pieces of gold," Hwang said. Among those most exciting are two common insertion sites identified in the study that each occurred in seven different p27-deficient animals, and rarely in the normal mice.

One of this pair is near a gene called Jundp2, which forms a protein known to interact with another cancer-causing gene called Jun. On its own, Jundp2 was not previously implicated in causing cancer. The other insertion is in a region of the X chromosome of the mouse, where genes have not yet been well characterized.

"Figuring out the identity of the genes near insertion sites was greatly facilitated by the recent completion of the mouse genome sequence," Clurman said. "The technique of insertional mutagenesis with the Moloney virus, combined with the resources of human and mouse genome sequences, allows one to start with one defined mutation-in our case, p27-and identify an enormous number of genes involved in cancer."

Clurman, also an assistant professor of medicine at the University of Washington, adds that the old approach to doing this was a tremendous fishing expedition. With the genome sequence, you have the direct address of a targeted region, allowing discovery of oncogenes involved in multiple tumors that would never have been found using old methods. This genomic approach has revealed that there are a lot more genes involved in these lymphomas, and probably in other cancers induced by these types of viruses, than had been known previously.

The team next plans to verify whether the genes they have identified are indeed activated in their set of experimentally-induced mouse lymphomas. If so, based on the similarity between the human and mouse genome, they can begin to examine whether human tumors contain hyperactivated forms of these genes as well. One or more of such cancer genes in a given pathway might serve as suitable targets for new cancer therapies.

"For example, we might not be able to develop a therapy that targets p27 directly," Clurman said. "But we may be able to target one of the collaborating proteins or pathways."

According to Clurman, the real power of this approach will be when he and others in the field amass a large collection of the components of many different cancer pathways.

"Other labs are looking for collaborators of genes associated with different cancer pathways," Clurman said. "A huge database will be built based of this information, which will allow us to compare the sets of genes altered in different kinds of tumors. Gene discovery is just our first step toward developing new approaches to treat human disease."