RNAi has proven to be so easy to use that many researchers who weren’t even aware of the gene-silencing technology until recently are taking advantage of it. Among them is Sharon Chang, a surgical resident at Dana-Farber Cancer Institute, who is using RNAi to uncover possible new roles for mammalian target of rapamycin, or mTOR, a kinase associated with cancer.
According to Chang, mTOR, which belongs to the phosphoinositide kinase-related kinase family, helps control the translation of proteins involved in cell growth and proliferation, as well as cell cycle progression. “It controls the translation of ribosomal machinery itself, and it also controls the translation of genes like myc and cyclin D1, which ultimately regulate cell cycle progression,” she told RNAi News.
Many cancers, Chang said, appear to stimulate a signaling pathway known as the PI3-kinase/Akt pathway. While this might be the result of a number of possible factors — including a mutation in or deletion of the PI3-kinase-opposing phosphatase PTEN; over-expression of Akt; or abnormal activation of other downstream elements — the end result is mTOR activation.
MTOR is regulated by the bacterial macrolide rapamycin, and “the dogma has been that rapamycin’s only target is mTOR,” Chang said. Given this, rapamycin and similarly acting agents have been scrutinized as attractive candidates for cancer drugs. “But the concern is that rapamycin may have targets other than mTOR,” she noted. “Furthermore, mTOR may have functions that aren’t necessarily sensitive to rapamycin.
“At this point, nobody really knows because the dogma has been that rapamycin equals mTOR inhibitor, and if you get an effect with rapamycin, everybody assumes that that’s because you’re inhibiting mTOR,” she said.
Contributing to the uncertainty was a paper appearing in Cancer Research in December by University of Pennsylvania researcher Amy Edinger, which presented data indicating that rapmycin’s inhibitory effects on mTOR are variable and that direct inhibitors of mTOR may have broader anticancer properties than rapamycin does.
The Edinger paper suggests that “there actually do seem to be distinct classes of mTOR activity, some of which are sensitive to rapamycin and some of which aren’t,” Chang said. “So the idea of the project [being undertaken at Dana Farber in the lab of Dirk Iglehart] is to see if using RNAi … to eliminate mTOR … would have different phenotypes than rapamycin treatment … in cultured breast cancer cell lines.”
Chang conceded that, going into this project, her knowledge of RNAi was limited. “I hadn’t even heard of [RNAi] until last July when I joined the [Iglehart] lab,” she said. But colleagues at Dana Farber have helped her overcome this issue.
“It’s kind of a collaborative effort,” Chang said. “We got the sequence from Bill Sellers, who’s been very active here using RNA interference, mostly for prostate cancer. He’s actually generated a library of good sequences.”
Additional assistance came from Andrew Kung, Chang said. “[He] happens to work right next door to us, and he’s the one who showed me how to cut the RNA into a hairpin and put that into a lentiviral vector.”
As for the RNAs themselves: “We bought the oligos from Dharmacon,” she said.
Chang said that some early work examining cell proliferation has been done, but she has encountered unforeseen difficulties that she is still trying to work past.
“What I’ve been finding with this lentiviral vector is that it’s pretty toxic to the cells,” she said. “So what I’m dealing with are issues of toxicity and the non-specific effects of having a whole bunch of hairpins floating around.” As a result, teasing out which effects are due to mTOR knockdown and which are due to the vectors or oligos has been challenging, despite comparisons with control vectors, she said.
Additionally, Chang said that timing has been a problem given the less-than-instantaneous effects of RNAi. “With rapamycin, you throw the drug on and it works,” she said. “But with RNAi, it’s a gradual process — you infect the cells, the virus has to integrate, the cells have to make the hairpins — and over time you’ll get the knockdown.”
Lastly, the very nature of the project has made the creation of stable cell lines problematic. “MTOR knockdown is unfavorable for cell viability, so over time the cells that have really good knockdown are selected out — they die,” Chang said. “The ones that survive, even though they may have moderate levels of knockdown, they’ve adapted. So, you’ve selected for cells that don’t care if mTOR is knocked down.”
Still, Chang is continuing with the project with the hopes that new details about mTOR and rapamycin might lead researchers to new cancer targets. “If you found that, for example, totally knocking down mTOR has more significant effects on growth inhibition than rapamycin does, that might lead you to target other areas of mTOR.”
Chang said that as it stands now, researchers aren’t completely clear on how rapamycin inhibits mTOR. “The thought is that if you see that getting rid of the protein altogether has very different effects than … rapamycin [introduction does], that might lead you to drugs that target the kinase domain, or other domains, directly.”
Additionally, should it be discovered the rapamycin affects targets other than mTOR, it could be possible to design a drug that “really, truly hits mTOR,” Chang said.