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RNAi, Meet P53: Cancer Research Uses Gene Silencing to Uncover Details of the P53 Pathway

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Cancer has proven to be a tempting target for RNA interference — and not just as a potential therapeutic approach. The gene-silencing technology is also being used to identify suitable targets for all varieties of cancer drugs, and one researcher at Cold Spring Harbor Laboratory believes that it could help answer some of the many questions still surrounding the widely researched tumor-suppressor gene p53.

Jack Zilfou, a postdoctoral fellow in Scott Lowe’s lab at CSHL, has like many cancer researchers been focusing on p53 because of its prevalence. The gene, he told RNAi News, is “impaired in over 50 percent of human cancers,” while some component of the gene’s pathway is likely mutated or abrogated in more than 90 percent of end-stage cancers.

Despite the level of attention that p53 receives from the oncology community, much remains unclear about the gene and its pathway, Zilfou said. So, after coming to CSHL, the home of RNAi pioneer Greg Hannon, “it [was] natural to dissect the pathway of this significant gene using this novel technology,” he said.

“Hopefully, if I can get a better understanding of [p53’s] role in … apoptosis or immortalization or genomic stability [for example], I can better understand how it works,” Zilfou said. “Once you identify … the genes that [p53] can turn on or turn off to get a specific phenotype, then you can use those as chemotherapeutic targets.”

To gain this better understanding of p53, Zilfou is taking advantage of three short-hairpin RNA constructs, developed in collaboration with Hannon’s lab, that are capable of knocking down p53 expression roughly 99 percent, 60 percent, and 25 percent, respectively, he said. These will be introduced into mouse embryo fibroblasts and the results will be analyzed.

By being able to control the level of p53 knock down, Zilfou expects to be able to see the different effects of the gene at various levels of expression. “When p53 is down almost 100 percent compared to a control, that residual p53 — that less than one percent that’s still there — can still activate target genes and can activate them substantially,” Zilfou said. “That’s what I think is amazing — that certain genes can still go up and other genes not.”

In earlier experiments, Zilfou said he has been able to “uncouple” immortalization from genomic stability. “Loss of p53 quickly … not only immortalizes cells, but causes massive genomic instability — in other words, [cells’] chromosome number is no longer diploid,” Zilfou said. “What I discovered is, if I can knock down p53 greater than … 99 percent, that small amount [remaining] is not enough to inhibit immortalization. But it is enough to maintain genomic stability — many of those cells have proper chromosome number similar to what the control has.”

Encouraged by this discovery, which “would not have been possible but for RNAi,” Zilfou said he wants to examine further what happens at the 99 percent knockdown level, as well as at the 60 percent and 25 percent levels.

“You can ask questions [like]: How much p53 is needed for this biological phenotype? How much p53 is needed to turn on this gene,” he said. “These are questions that can only be asked with this level of control due to the development of RNA interference technology.”

Previously, “attempts had been made to try to over-express p53 in cells … but then you have over-expression artifacts, the protein may not be modified property, [or] it may not be localized properly,” Zilfou added. “By using RNAi and knocking it down endogenously, [we have] a way of tightly regulating [p53].”

Zilfou said that his work could help give researchers a sneak peek of sorts into tumor progression by creating a situation that “may [resemble] certain early events in tumorogenesis.” Additionally, he expects that studies of what genes are turned on and off in cell lines with the different levels of p53 expression will help uncover “the players in a [particular] biological phenotype.”

Down the road, Zilfou said that he sees this project carrying to the realm of in vivo experimentation. “As these cell-based experiments begin to reveal certain elements of the p53 pathway that are significant in processes like immortalization and genomic stability … we can go back and try to better understand what those targets are and what they do” in animal models, he said.

As for RNAi as a therapeutic modality for cancer, Zilfou said he sees this as a very real possibility — if, of course, the problem of delivery can be adequately solved.

— DM

 

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