NEW STRATEGY EMPLOYS RNA INTERFERENCE TO IDENTIFY NOVEL CANCER SUPPRESSOR GENE

UMass Medical School investigators define new method for screening for genes that suppress metastasis in melanoma; genome-wide screening approach systematically narrows potential targets for drug development.


WORCESTER, Mass.—Metastasis—the spreading of cancer from a primary tumor to other places in the body—is the major cause of deaths from solid tumor cancers; and the biological processes that advance metastasis and cause tumors to spread are complex, controlled by multiple genes.  While numerous genes that promote metastasis have been isolated, genes that suppress a cancer’s ability to metastasize have traditionally proven more difficult to identify.

But scientists at the University of Massachusetts Medical School in the lab of Howard Hughes Medical Institute investigator Michael R. Green, MD, PhD, the Lambi and Sarah Adams Chair in Genetic Research and director of the Program in Gene Function and Expression at UMMS, have developed a systematic method for screening the genomes of cancer cells to detect likely metastasis suppressors.

In their paper in the November 1, 2008 issue of the scientific journal Genes & Development (Gobeil, S., Zhu, X., Doillon, C.J. and Green, M.R. (2008) “A genome-wide shRNA screen identifies Gas1 as a novel melanoma metastasis suppressor gene.” 22(21) 2932-2940), Green and colleagues used two techniques—a three-dimensional cell culture system in conjunction with genome-wide RNA interference screening—to identify novel genes that suppress metastasis in a common and deadly form of cancer called melanoma.  Both the discovery of at least one previously unknown genetic pathway that appears to suppress the spread of cancer, and the method of using cell cultures suspended in a three-dimensional system that abets identification of aggressive metastatic cancer cell colonies, are exciting advances at the frontier of cancer biology and genetic expression.

“Although many genes have been identified that promote metastasis, a relatively small number of metastasis suppressor genes have been documented,” according to the authors.  “This is due at least in part to a lack of experimental approaches for the systematic identification of genes that specifically inhibit metastasis.”

Green et al identified a batch of 22 genes that, when “knocked down” or silenced, increases metastasis without impacting primary tumor growth.  Focusing on one of the genes, Gas1, subsequent experiments demonstrated that this gene had all of the expected properties of a melanoma metastasis suppressor gene:  suppression of metastasis in mice; promotion of programmed cell death in cells that have spread to secondary sites; and down-regulation in other experimental cell lines, including human melanoma-derived lines and melanoma tumors.

Further experiments are needed to examine the other 21 genes, but Green thinks they are highly likely to be suppressors of metastasis for a variety of cancers.

“Metastasis is an important part of cancer biology, but it is a complex process and a tough problem to study,” says Green. “The most exciting part of the paper is not necessarily that we have found this one metastasis suppressor but that we’ve developed a general approach that can be used to find others—and we can do it with any type of cancer cell.”

Like tumor-suppressor genes, those that suppress metastasis are most evident when they are damaged or altered in ways that disrupt their protective function. The first metastasis-suppressors were unearthed in the late 1980s. Reduced activity of some of those genes has been found in cancers through microarray studies that measure gene activity in cells. It is still far from clear, however, how the loss of such a gene, or genes, enables a cancer cell to do the things it must when it metastasizes: that is, operate apart from the original tumor; invade normal tissues; survive a long-distance journey through the blood stream; and establish a thriving colony in a target organ.

In devising a functional screen for metastasis-suppressing genes, Green and his colleagues, including first author Stephane Gobeil, exploited two recent technological advances – RNA interference and three-dimensional cell-culture systems.

Traditional cells cultured in sheets on flat plastic dishes fall short of replicating the natural surroundings of cells in the body, with their neighboring cells, fibrous layers, membranes, and adhesion proteins. Newly developed three-dimensional systems are a boon for studying metastasis, says Green. “They are a system that allows you to mimic early events in the metastasis cascade,” he says.

In Green’s laboratory, researchers inserted balls of about 1 million cells into collagen, forming a plug that is then coated with an artificial matrix that mimics a cellular basement membrane. Next, the coated plug is embedded in a fibrin matrix within a standard Petri dish. In metastasis assays, cancer cells can be observed as they break off and migrate away from the original cell mass in all directions.

For the experiments, Green chose two mouse melanoma cell lines: One, B16-F0, is only weakly metastatic, while B16-F10 has a high potential for metastasis. The investigators used RNA interference tools – so-called small hairpin RNAs (shRNAs) – to lower the expression of genes in the nucleus of each type of cancer cell. The screen was designed to select for melanoma cells carrying shRNAs that silenced metastasis-suppressing genes, allowing the cells to form satellite colonies. The DNA of those cells was extracted and sequencing identified the shRNAs responsible – and, in turn, the genes themselves.

The screen yielded 78 genes that promoted colony formation from the weakly metastatic B16-F0 line, suggesting their knockdown removed a suppressive function. Next, the scientists administered those 78 shRNAs to the melanoma cells and injected them into the tail veins of mice. Fourteen days later, they examined the rodents’ lungs for metastases, and found significant numbers of metastases in mice receiving 22 of the 78 gene-silencing RNAs. Among the 22 were several genes known to play a role in signal transduction, cell cycle regulation, or metabolism/energy pathways.

One gene of interest, Gas1 (growth arrest-specific 1), is known to govern cell growth. It was markedly down-regulated in the metastatic melanoma cell, said Green. To test this candidate metastasis-suppressor, the researchers injected melanoma cells in which Gas1 expression had been reduced into the footpads of mice – a more rigorous metastasis assay than the previous tail-vein injection. Those cells generated significantly more metastases in the lungs than cells with normal Gas1 activity.

The experiments by Green and his colleagues suggest that the Gas1 gene normally squelches metastasis by killing migrating cancer cells after they have reached their destination. It does so by turning on the programmed cell death, or apoptosis, machinery in melanoma cells, causing them to self destruct. When Gas1 is damaged, the tumor cells can escape their death sentence and live to form metastatic colonies of cells.

The scientists are eager to expand their Gas1 studies to other cancers; it has been previously found to be down-regulated in breast and prostate cancer metastases as well. In addition, Green says, they have plenty of work to do in putting the remaining 21 candidate genes through their paces to see if they are true metastasis-suppressors.

Green, who became a Hughes investigator in 1994, is one of five HHMI investigators at UMass Medical School. The other four are: Roger J. Davis, PhD, the H. Arthur Smith Chair in Cancer Research and professor of molecular medicine (1990); Craig C. Mello, PhD, the Blais University Chair in Molecular Medicine and professor of molecular medicine (2000); Melissa J. Moore, PhD, professor of biochemistry & molecular pharmacology (1997); and Phillip D. Zamore, PhD, the Gretchen Stone Cook Chair in Biomedical Sciences and professor of biochemistry & molecular pharmacology (2008). Their laboratories are funded by HHMI while they retain their faculty appointments at the Medical School and pursue their research.


About the University of Massachusetts Medical School
The University of Massachusetts Medical School has built a reputation as a world-class research institution, consistently producing noteworthy advances in clinical and basic research.  The Medical School attracts more than $193 million in research funding annually, 80 percent of which comes from federal funding sources. The work of UMMS researcher Craig Mello, PhD, an investigator of the prestigious Howard Hughes Medical Institute (HHMI), and his colleague Andrew Fire, PhD, then of the Carnegie Institution of Washington, toward the discovery of RNA interference was awarded the 2006 Nobel Prize in Physiology or Medicine and has spawned a new and promising field of research, the global impact of which may prove astounding. UMMS is the academic partner of UMass Memorial Health Care, the largest health care provider in Central Massachusetts. For more information, visit www.umassmed.edu.