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RNA Interference Genetic Screen Suggests New Targets for Cancer Therapies

Researchers at the National Cancer Institute (NCI), part of the National Institutes of Health, have developed a new method to identify genes that keep cancer cells active and that could be potential targets of anticancer therapies.

The method uses RNA interference (RNAi), a technology for silencing genes, to screen cancer cells for genes that, when silenced, cause cancer cells to die or stop dividing. These genes are essential for the survival of cancer cells and represent potential therapeutic targets, but they might not contain mutations or other alterations typically associated with the disease.

“This method could be used to identify a new class of oncogenes beyond the ones traditionally identified as mutated or otherwise deregulated in cancer,” said lead researcher Louis M. Staudt, M.D., Ph.D., of NCI’s Center for Cancer Research (CCR). The method is described in a study to be published online in Nature* on March 29.

“The traditional way of approaching cancer biology is to identify genes in cancer cells altered by mutations or in their functions,” explained Staudt. “This method identifies additional genes that are not necessarily mutated or altered but that are nonetheless required for the cancer cell’s survival. These additional genes could provide a range of therapeutic targets beyond the small set of genes we have already identified.”

The researchers used the method, technically called a loss-of-function RNA interference genetic screen, to identify three genes not previously linked to cancer. These genes turn on a cellular process, or pathway, that is continuously activated in a type of lymphoma cell. Lymphoma is a cancer of the lymph nodes. The genes could become targets of therapies for a type of lymphoma called activated B cell-like diffuse large B-cell lymphoma (DLBCL).

“The genetic screen revealed a new mechanism in this lymphoma that we didn’t know about before,” said Staudt. “More broadly, there is an opportunity to apply this genetic screen to all types of cancer in order to create a new classification of the disease based not on cancer type, but on which pathways inside a cancer cell are critically required for its proliferation or survival.”

“This type of functional classification is critical because what we need to know most about a cancer cell is which pathways should be targeted for any particular cancer.” he continued. “We call it an Achilles heel genetic screen because it identifies the pathways in the cancer cell that are most vulnerable to attack.”

The screen is similar to those used to mutate and to study genes in laboratory animals. In this case, RNAi is employed to reduce the activity of a specific gene in a living cancer cell, and then to see whether the cell can survive. RNA interference alters the levels of RNA in a cell, thereby reducing the amount of protein produced by the targeted gene.

The technological advance made by Staudt’s team was to create an on/off switch that allowed them to activate the production of specific short hairpin interfering RNA molecules, or shRNAs, once the genetic code for the shRNA was delivered into a cancer cell using a modified virus. Until now, experimentally delivering certain shRNAs into cells could kill the cells immediately.

“The inducible shRNA virus allowed researchers to infect a cancer cell while shRNA production is in the off mode,” explained Staudt. “When we added drugs that induced the expression of the shRNA, and if the gene targeted by the shRNA was essential, then the cell died.”

As a demonstration of this technique, the researchers screened 2,500 genes in two types of diffuse large B-cell lymphoma cells. Their previous research had suggested that the NF-kB signaling pathway — which is involved in regulating the expression of a large number of genes — is critical in the activated B cell-like (ABC) type, but not in the germinal center B cell-like (GCB) type. These lymphomas have very different survival rates and patterns of gene activity.

“Our hypothesis was that we should find different genes that are required for the proliferation or survival of the lymphomas because these are very different diseases clinically,” says Vu N. Ngo, Ph.D., also of CCR, who led the experiment.

In the experiment, the researchers created shRNAs for 2,500 human genes. They grew cell cultures containing the two types of lymphoma cells and delivered a single shRNA to each cell. After a drug was added to induce the expression of shRNAs, the researchers used DNA microarray technology and molecular tags attached to the shRNAs to identify genes that were essential for cell survival and growth.

The experiment confirmed previous research findings that genes involved in the NF-kB signaling pathway are essential for the survival or proliferation of ABC DLBCL cells, but not cells of the GCB type. The screen also identified three other genes that are essential for the survival of the ABC-type cells only. One of these three genes is called CARD11, and it appears to interact with two other genes, MALT1 and BCL10, to activate a pathway required for the survival of ABC-type lymphoma cells.

The researchers plan to expand the screens to include cell cultures representing all types of human lymphomas and eventually all types of human cancers. The Achilles heel genetic screen is complementary to NCI-led efforts to sequence human cancer genomes, said Staudt, because identifying critical pathways in cancer cells will help focus the search for relevant genetic mutations.

Staudt will present findings from the study at the American Association of Cancer Research annual meeting in Washington, D.C., on April 3, 2006.

For more information about cancer, please visit the NCI Web site at http://www.cancer.gov, or call NCI’s Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).

The National Institutes of Health (NIH) — The Nation's Medical Research Agency — includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. It is the primary federal agency for conducting and supporting basic, clinical and translational medical research, and it investigates the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit http://www.nih.gov.


* A loss-of-function RNA interference screen for molecular targets in cancer. Ngo VN, Davis RE, Laurence L, Yu X, Zhao H, Lenz G, Lloyd L, Sandeep D, Yang L, Powell J, and Staudt LM. Nature, online March 29, 2006.
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