Two groups of researchers this week separately published data showing that the p53 tumor-suppressor gene directly targets members of the evolutionarily conserved miR-34 family of microRNAs, which is known to suppress cell proliferation.
The findings, which appeared in the online editions of Nature and Molecular Cell, suggest that this small non-coding RNA family may be a key component in the p53 tumor-suppressor network, which controls cellular responses to signals such as DNA damage and oncogene activation.
In the Nature paper, researchers from Cold Spring Harbor Laboratory, Rosetta Inpharmatics, and Applied Biosystems describe how three members of the miR-34 family are seemingly employed by p53.
“A global decrease in … miRNA levels is often observed in human cancers, indicating that small RNAs may have an intrinsic function in tumor suppression,” the paper’s authors wrote. To identify miRNA components of tumor-suppressor pathways, they examined miRNA expression profiles in both normal and p53-deficient mouse embryonic fibroblasts.
The researchers measured the expression of a panel of 145 mouse miRNAs in wild-type or p53-deficient MEFs that ectopically express various oncogenes. They found that the expression of three miRNAs — miR-34a, miR-34b, and miR-34c — was “precisely correlated” with p53 status, the authors report. “This raised the possibility that miR-34 genes might be regulated by p53,” they wrote.
Additional experiments confirmed that genes encoding the miR-34 family members are direct transcriptional targets of p53. Additionally, ectopic expression of miR-34a, miR-34b, and miR-34c induced cell-cycle arrest in primary and tumor-derived cell lines, which the authors note is “consistent with the observed ability of miR-34 to down-regulate a program of genes promoting cell cycle progression.”
The researchers note that the miR-34 family of miRNAs is a “direct target of p53 that possess anti-proliferative potential” and suggest that ”it is likely that miR-34s mediate this response through additive or synergistic effects of multiple targets, because many components of the cell cycle machinery are affected after the manipulation of miR-34 levels.”
The Nature authors also noted that while numerous p53 targets have been identified in mammals, “very few” are evolutionarily conserved in Drosophila and C. elegans even though they retain homologues of the p53 pathway.
Furthermore, miR-34 is “one of only 18 mammalian miRNA families that are also present in flies and worms,” which the researchers said raises the possibility that “the link between p53 and this non-coding RNA target may have arisen early in the evolution of the p53 network and may be important in p53 function in diverse species.”
Meanwhile, in Molecular Cell, investigators from Johns Hopkins University School of Medicine reported that miR-34a appears to modulate and fine-tune the gene-expression program triggered by p53.
After conducting global miRNA expression analyses in p53 wild-type colon cancer cells and an isogenic cell line in which both alleles of p53 were inactivated, the researchers identified miR-34a as the miRNA exhibiting the greatest level of expression change when the cells were treated with a DNA-damaging drug known to induce p53.
Experiments then confirmed miR-34a was a direct transcriptional target of p53, and the phenotypic effects of the miRNA were examined.
The team transiently transfected miR-34a into the wild-type and p53-deficient colon cancer cells, then measured apoptotic cell death. They found that transfection of the wild-type cells resulted in cell death, while apoptosis was “substantially decreased,” but not eliminated after transfection in the p53-deficient cells.
”These data suggest that miR-34a participates in the apoptotic program triggered by p53 activation,” the authors wrote.
“miR-34 is one of only 18 mammalian miRNA families that are also present in flies and worms … [raising] the possibility that the link between p53 and this non-coding RNA target may have arisen early in the evolution of the p53 network and may be important in p53 function in diverse species.”
Additional experimentation in pancreatic cancer cells, along with the results of previous studies by other groups indicated that the loss of miR-34a is a “frequent event in diverse cancer subtypes and raises the possibility the miR-34a loss of function contributes to cancer pathogenesis,” the authors added.
To study the effects of miR-34a induction on gene expression, the researchers generated colon cancer cells with enforced miR-34a expression. These cells, they wrote, “showed a dramatically altered gene expression profile” in which 532 transcripts were up-regulated and 681 were down-regulated.
However, “the majority of gene expression changes are likely an indirect consequence of enforced miR-34a expression,” the authors noted.
“Our results show that p53 directly activates expression of miR-34a and potentially other miRNAs,” the authors wrote. “Expression of miR-34a is sufficient to induce apoptosis through p53-dependent and -independent mechanisms. Moreover, miR-34a induction leads to dramatic reprogramming of gene expression.
“Much like the known set of p53-regulated genes, miR-34a-responsive genes are highly enriched for those that regulate cell-cycle progression, cellular proliferation, apoptosis, DNA repair, and angiogenesis,” they added. “Consistent with this observation, we show that reduced expression of miR-34a is a very frequent feature of pancreatic cancer cells. Together, these findings suggest an important role for miR-34a in mediating p53 tumor suppressor function.”