Although RNAi’s future as a therapeutic is uncertain, the technology has been wildly successful as a means for determining gene function. Just about every major drugmaker uses RNAi to support its drug discovery and target validation efforts, and Aventis is no exception.
Speaking at IBC’s 2nd International RNAi conference in Boston this week, Dorre Grueneberg, RNAi platform head at the Aventis Cambridge Genomics Center, discussed how the company has been using RNAi to identify genes that affect the efficacy of its top-selling breast and lung cancer drug Taxotere.
Grueneberg said that the company has developed screens that “allowed us to identify siRNAs that make cells either more resistant or sensitive to Taxotere treatment.” She noted that the screens are, however, biased, given that some of the siRNAs the company looked at knocked down genes that had been already identified as being up-regulated in treatment-resistant breast cancer tissues.
Describing the process of making the screens, Grueneberg said that “on day one, we place HCT116 cells in a 96-well format.” The next day, the siRNAs are transfected into the cells. On day three, the media is removed and different concentrations of Taxotere are added.
“On the fourth day, we remove the Taxotere and add fresh media,” Grueneberg said. “And on the sixth day, we remove the media and add WST-1,” which is a colorimetric enzymatic assay for the quantification of cell proliferation and viability.
“What we do is analyze the data, calculating the kill curves,” she said. “As you increase the Taxotere concentration, you decrease the cell viability. And so, if something is resistant to treatment, it shifts the curve [in one] direction. If an siRNA confers sensitivity, it shifts it [in the other].”
Of the 52 siRNAs the company has already screened — there are about 50 more still to be examined — “the majority had no effect,” Grueneberg said. However, five were found to confer sensitivity to Taxotere and two were found to confer resistance. She declined to name the siRNAs’ target genes.
With these siRNA data under their belt, and more coming as additional siRNAs are examined, Grueneberg said that she and colleagues at the Aventis Pharmaceu- tical Cambridge Genomics Center are generating stable knockdown cell lines “and then studying the effects of Taxotere on cell killing.”
To generate the cell lines, she said, the researchers infect HCT116 cells with short hairpin RNAs against the genes found to cause Taxotere sensitivity/resistance. Once the knockdown effect has been confirmed, the cell lines are treated with Taxotere. The drug is then removed, and the cells are treated with WST-1.
Grueneberg said that results thus far are positive, with the cell lines derived from the siRNAs that confer Taxotere sensitivity also conferring sensitivity and vice versa for the siRNAs conferring drug resistance.
Aventis, Grueneberg said, hopes to use the identification of the Taxotere sensitivity-conferring siRNAs as a springboard to developing small molecule drugs that could be used in conjunction with the drug, enhancing its efficacy. As for the resistance-conferring siRNAs, she said that these may help the company develop diagnostics to weed out patients with a genetic makeup that would make them poor candidates for drug treatment.
Grueneberg recently co-authored a paper describing the development of a screen in human cells that identified UBF2 as an RNA polymerase II transcription factor enhancing the beta-catenin signaling pathway, which plays a role in driving the malignant transformation of cancerous epithelial cells. (See Molecular and Cellular Biology, June 2003; 23: 11, 3936-3950.)
In that paper, she and colleagues wrote that coimmunosuppression experiments revealed that UBF2 associates with LEF-1 and potentiates transcriptional activities stimulated by LEF-1/beta-catenin from a synthetic promoter with multimerized LEF/TCF binding sites and a natural cyclin D1 promoter with consensus LEF/TCF binding sites.
Additionally, downregulating UBF expression using RNA interference reduced transcriptional activation of a beta-catenin-LEF/TCF-response promoter by means of overexpressed beta-catenin, they wrote, adding that this further implicated UBF as a transcriptional enhancer of the beta-catenin pathway.
—DM