Journal of the National Cancer Institute Advance Access originally published online on November 13, 2007
JNCI Journal of the National Cancer Institute 2007 99(22):1664-1665; doi:10.1093/jnci/djm242
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© Oxford University Press 2007.
NEWS |
BIG JOB FOR LITTLE RNAs
MicroRNAs Found Cavorting With p53
The tumor suppressor protein p53 has long held the spotlight as master of ceremonies in the meteoric rise of malignancies. Now, a class of small RNAs that has been waiting in the wings appears poised to take center stage—or at least dance backup.
Over the last several years, more and more of the small RNAs called microRNAs have been uncovered performing a variety of duties in cancer (see J Natl Cancer Inst 2006;98:885–7). Now, several research groups have unearthed microRNAs working with one of the most infamous players in cancer biology: tumor suppressor p53.
The finding comes as no surprise, given the number of microRNAs being found in cells. "We expected that some of the targets of such a prominent transcription factor as p53 would be microRNAs," says molecular biologist Guido Bommer, M.D., at the University of Michigan School of Public Health in Ann Arbor.
The past year has seen a burst of studies that link p53 to a family of microRNAs called miR-34. Work from many different laboratories revealed that this set of three microRNAs are involved in the cell cycle and apoptosis, two cellular systems that cancer perturbs. p53 activates miR-34, and miR-34 slows or stops the production of other proteins. "There are about five to 10 experimentally validated targets of miR-34," Bommer says.
The three small RNAs that make up the miR-34 family reside in different places in the genome: miR-34a resides on one chromosome, and miR-34b and miR-34c on a different one. MiR-34b and -34c are produced as one copy and then trimmed down (the combined form is sometimes called miR-34bc). The three RNAs are almost identical, but different kinds of cells produce different amounts of them.
MicroRNAs can prevent proteins from being produced by virtue of their nucleotide sequence. A short sequence complements a sequence found on the target's messenger RNA, and those two RNAs stick together. That link either blocks the message from being copied into protein by the protein-making machinery of the cell or it degrades the message altogether.
To find microRNAs that were controlled by p53 in the first place, cancer biologist Joshua Mendell, M.D., Ph.D., of Johns Hopkins University School of Medicine and his colleagues used colon cancer cells that had been engineered to lack p53. The team damaged the cells’ DNA and monitored which microRNAs increased production. By comparing results from the cells with and without p53, the researchers found eight microRNAs ramped up by p53. "MiR-34a was the most robustly induced," Mendell says.
Molecular biologist Gregory Hannon, Ph.D., of Cold Spring Harbor Laboratory in New York and colleagues performed similar experiments in embryonic mouse cells. In addition to comparing microRNAs in cells that contain p53 versus those that do not, the team curbed the production of p53 and then let the cells produce p53 again. The production of miR-34 increased in parallel with that of p53 in the cells that resumed making the tumor suppressor protein.
But just because both molecules arise together does not necessarily mean that they work together. To determine whether p53 directly works with miR-34, Mendell's team mutated the nucleotide sequence in miR-34 that was suspected to interact with p53 and found that the cells could no longer increase their production of the microRNA. Hannon's group found that when they used antibodies to fish out p53 from mashed-up cells, miR-34a and miR-34bc came along for the ride. Together, these experiments showed that the protein and the RNA molecule work side by side.
Mendell's group then tested what happens when miR-34 ramps up in the absence of any DNA damage. They infected colon cancer cells with copies of the microRNA, and about a quarter of the cells underwent apoptosis, or programmed cell death. However, when they repeated the experiment in the cells without p53, only 10% of the cells did so. "That tells us that there are multiple mechanisms by which apoptosis happens," Mendell says.
A second group, led by Moshe Oren, Ph.D., of the Weizmann Institute of Science in Israel, also demonstrated miR-34a's role in apoptosis: Inactivating miR-34a prevented p53 from causing cultured cells to self-terminate. And increasing the amount of the microRNA increased p53-induced apoptosis, mirroring Mendell's result.
Bommer found one way in which miR-34a could induce apoptosis. The protein Bcl2 normally protects a cell from undergoing programmed cell death. When he mixed molecules carrying the regulatory region of Bcl2 RNA and miR-34a RNA, the micro molecule prevented the Bcl2 RNA from being converted into a protein. When miR-34a levels go up in a cell, Bommer suggests, they could turn off Bcl2, allowing cells to kill themselves.
In addition to causing apoptosis, the microRNA can simply stop a cell from growing. Cultured colon cancer cells normally produce small amounts of miR-34a. Bommer's group overproduced miR-34a in those cells and found that the cells quit growing. This result suggested that miR-34a controls some molecular players in the cell cycle.
Support for this result came from Hannon's laboratory. His team produced the miR-34 molecules in cultured cells of four tumors and looked for proteins whose production fell over the next 24 hours. He further investigated three proteins as possible targets of miR-34, two of which are involved in the cell cycle: cyclin E2; cyclin-dependent kinase 4; and a protein involved in liver cell growth, hepatocyte growth factor receptor.
Hannon then asked whether miR-34 carries p53's cease-and-desist signal to the targets. MiR-34 sticks to a particular six-nucleotide sequence in messenger RNA molecules, and Hannon's group mutated that sequence in their three proteins of interest. The mutation allowed the kinase and the growth factor receptor to continue to be produced even in the presence of miR-34a, and cyclin E2 recovered to about 80%. These results suggest that p53 uses miR-34 to turn off some genes.
Moving from culture to real life, Oren's team showed that p53 activates miR-34 in animals: Irradiating mice increases their production of miR-34a. Furthermore, enhanced production of miR-34a in animals can actually protect against cancer. Hannon injected mice with a type of liver cancer cell whose p53 is temporarily suppressed, allowing the cells to grow malignantly. Once the suppression of p53 was lifted, the cells pumped out high levels of the miR-34s. And the tumors stopped growing, strongly suggesting that miR-34a can function as a tumor suppressor similar to p53.
While miR-34a seems to be stealing the spotlight, miR-34b and -34c also have roles, depending on the tissue type. Bommer examined levels of miR-34 in a variety of tissues and found that the lung had the highest level of miR-34bc. Delving into lung cancer, he found that six of 14 lung tumor types had lost much of their miR-34bc. In these samples, miR-34a was not consistently up or down, he says.
Many questions remain to be answered about miR-34. For example, Mendell says that how miR-34 works its magic is not clear. "The microRNA could be regulating one target or modulating expression of many transcripts to a small degree," he says. And with some pancreatic cancer cells, the cells retain p53 instead of losing it and get rid of miR-34 instead. "It's possible that miR-34 is a tumor suppressor," he says.
Bommer's group is currently trying to knock out the miR-34 genes in mice to see what happens in those animals. Along with all the tissue culture results, such work will help researchers understand how big of a role the tiny molecule plays in the huge scourge that is cancer.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||