1. Regulation of the cyclin dependent kinase inhibitor p27 in glioblastoma
- Author
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Gillies, Jana K
- Subjects
Chemistry, Biochemistry ,Biology, Molecular - Abstract
Glioblastoma Multiforme (GBM) is the most common and most malignant brain tumour. The cyclin dependant kinase inhibitor p27 is commonly inactivated in this and many other cancers. p27 is an important negative regulator of the cell cycle which acts by inhibiting cyclinE/cdk2 and cyclinA/cdk2 complexes. We identify PKCiota, a pro-proliferative kinase which is activated by phosphoinositide 3 kinase (PI3K), as a regulator of p27 in GBM. siRNA-mediated depletion of PKCiota in U87MG cells resulted in an increase in p27 protein levels, and p27 levels are extremely high in PKCiota-null mouse embryonic fibroblasts. The mechanism by which PKCiota regulates p27 is unclear, although it occurs at the post-transcriptional level, as QRTPCR analysis showed that PKCiota depletion did not alter p27 mRNA levels. PKB is a known regulator of p27, but we show that PKCiota does not regulate p27 through modulation of PKB activity. MEK/ERK signaling is also implicated in the regulation of p27. The combined inhibition of PKCiota and MEK resulted in an increase in p27 levels which was greater than the added effects of inhibiting either kinase alone, suggesting some overlap in the function of these two pathways. Additionally, we show that p27 is regulated by miRNAs, which are 20-25 nucleotide RNAs that repress gene expression by post-transcriptional silencing. In eukaryotes, this occurs predominantly at the level of translational inhibition and is mediated by defined sequences in the 3'UTRs of target genes. miRNAs are processed by dicer, and therefore the deletion of dicer abrogates miRNA expression. We show that siRNA-mediated depletion of dicer increases p27 levels and halts the cell cycle at G1 phase. The miRNA target site prediction program Targetscan predicted 2 binding sites for miR-221/222 in the p27 3'UTR. Transfection of glioblastoma cells with inhibitors of miR-221 and/or miR-222 resulted in an increase in p27 protein levels. These results were corroborated by luciferase reporter assays in which dicer depletion or miR-221 inhibition increased activity from a luciferase reporter gene located immediately upstream of the p27 3'UTR. Site-directed mutagenesis of the predicted miR-221 binding sites in the reporter construct increased luciferase activity and abrogated the effect of miR-221 inhibition. Therefore, p27 is regulated by miR-221. We also show that PKCiota and MEK alter p27 levels via an element in the 3'UTR. These results are consistent with a model in which oncogenic signaling pathways increase miR-221 expression to decrease translation of p27.
- Published
- 2013
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