Pathway analysis once meant a lot of by-guess-and-by-golly work. But thanks to genome-wide RNAi screens, the art of elucidating pathways is rapidly becoming a high-throughput science. Interference technology has now become one of the most promising tools for deciphering signaling cascades and for finding new genes involved in them.
“RNAi large-scale screens [put] us in a position to ask questions that we simply couldn’t ask, let’s say, five years ago,” says Rene Bernards, cancer researcher and CSO of Agendia. In his own studies, Bernards uses large-scale screens to study numerous pathways that range from p53 in cancer to NF-kB signaling, cell migration, and protein localization in subcellular compartments. His 2004 Nature paper was one of the first to use large-scale loss-of-function genetic screens in mammalian cells, which identified five new modulators of p53-dependent cell-cycle arrest.
Certain components of specific cancer-relevant pathways are known, he says, “but the fact that we know, let’s say, 10 components of a particular pathway doesn’t mean that is all there is. RNAi lets you now, in an unbiased fashion, ask, of all the genes in the human genome, which players act in this pathway? It gives a complete survey of the genes that act in such a pathway.”
Bill Hahn, a member of the RNAi Consortium at the Broad Institute, is also making use of large-scale screens to find genes that contribute to cancer phenotypes. One way is to “screen collections of RNA targeting either parts of the genome or the whole genome in a specific assay, say proliferation, [and then] look for all the genes that might regulate proliferation or mitosis or some other assay,” Hahn says. “Hopefully … after you screen enough of a pathway … you’ll find several members of the pathway, all of which regulate the same process.”
Despite the prevalence of using RNAi screens to decipher cancer pathways, they can and are readily being used for almost any elucidation work. Says Harvard’s Steve Elledge, “It’s being used for everything, genetically.” In his own work, Elledge has partnered with Greg Hannon at Cold Spring Harbor Laboratory to build a vector-based shRNA library, which covers the mouse and human genomes. Some of his recent papers have used RNAi screens to discover tumor suppressor REST, spindle checkpoint proteins USP44 and TAO1, and Myc-regulator, USP28.
The Broad, along with MIT and Harvard, is using its own lentiviral vector-based library, which covers 16,000 genes in both mouse and human, to do large-scale knockout screens that explore immune response pathways in a collaborative project called Immune Circuits.
Though a powerful tool, RNAi is not foolproof. Off-target effects, reducing signal-to-noise ratio, and target verification in large-scale screens are major points of contention to the technology’s ultimately becoming a reliable genomic tool. Part of the RNAi Consortium’s work at the moment is updating its library, both by expanding it and by measuring the knockdown performance of the constructs. Consortium members would like to obtain coverage of two or more hairpins with experimentally validated strong knockdown for each target gene in order to be better able to interpret the primary screening data, says Dave Root, director of the consortium.
“RNAi is not perfect in the sense that there are off-target effects and you have to be very careful about interpreting screens,” Hahn says. “And at this point, nobody’s library, including ours, is completely validated to see which vectors knock down a gene and how much they knock down a gene. You have to use RNAi knowing its limits and its capabilities and work within those.”
Adds Hannon, “In the end, this really is a genetic tool where the screen itself generates candidates and that one must then go verify those candidates with further work.”
Integrative genomic approaches identify IKBKE as a breast cancer oncogene
Research led by Bill Hahn and published in the June 15, 2007, issue of Cell used genome-wide screens to identify a kinase oncogene, IKBKE, which acts downstream of AKT and is amplified in many human breast cancers. Through introducing mutant alleles into immortalized human kidney epithelial cells, they found that a mutant myr-AKT along with another allele activated the P13K pathway, which was known to be activated by Ras and induce tumor growth. They used a similar assay to identify IKBKE, a kinase that also stimulates P13K-induced transformation. Using high-density SNP arrays, they found copy number gains associated with the IKBKE gene that correlated with over-expression in tumor cells. They then screened 6,144 shRNA constructs against 1,200 genes and found that three out of five targeting knockdown of IKBKE reduced cell proliferation in MCF-7 breast cancer cell lines. Further study showed that IKBKE expression activated the NFkB pathway.
A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression
A leader in the field of Drosophila RNAi screens, Norbert Perrimon at Harvard Medical School continues pioneering work in the field. A recent paper, published in the May 29, 2007, issue of PNAS, used a genome-wide RNAi screen to discover genes that negatively regulate E2F activity independently of Rb. E2F is known to be a key regulator of cell proliferation and differentiation, but thought to be repressed by the Rb tumor suppressor. In this study, Perrimon used a Drosophila reporter-based assay by creating an E2F-responsive firefly luciferase reporter that could monitor endogenous E2F activity. The team found that knocking down the Domino chromatin remodeling complex and the Polycomb Group (PcG) protein-like fly tumor suppressor L3mbt and related CG16975/dSfmbt activated the reporter. In follow-up functional assays, they found that these factors are physically recruited to promoters and are required in order to repress the target E2F genes.
A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signaling
Harvard’s Steve Elledge led a research effort that used a genome-wide shRNA screen to identify additional human spindle-checkpoint proteins. The work was published in the May 2007 issue of Nature Cell Biology. Previous genetic screens in yeast have identified several spindle checkpoint proteins, including Mad1, Mad2, Bub1, Bub3, BubR1, and Mps1, but the human inventory is far from complete. To this end, Elledge screened a total of 2,533 shRNAs against 780 genes targeting human kinases and phosphatases, and then assayed the knockdowns after taxol treatment to see which had bypassed checkpoint arrest. The group identified a microtubule affinity-regulating kinase kinase, TAO1, as an important regulator. Using further functional assays and partial siRNA gene knockdown, they found that TAO1 is an interactive protein that has two functions, regulating both microtubule dynamics and checkpoint signaling.
The ubiquitin-specific protease USP28 is required for MYC stability
Bernards, Elledge, and Martin Eilers of the University of Marburg, Germany, used a barcode shRNA screen to identify — among other genes — USP28, a ubiquitin-specific protease that is required for MYC stability in human tumor cells. Their work was published in July 2007 issue of Nature Cell Biology. As a proto-oncogene, MYC is thought to contribute to tumor cell proliferation at enhanced levels. Using a pooled barcode microarray RNAi screen, the scientists infected cells with a library of 23,742 shRNA vectors targeting 7,914 genes. From this, they isolated an initial 91 genes that play a role in MYC function, and through individual shRNA knockdown tests, identified USP28 as essential for MYC stability. Additional functional assays showed that USP28 binds to MYC through interacting with FBW7a, an F-box protein that is part of an SCF-type ubiquitin ligase.