Abstract A61: MiR-101 as a molecular switch for sequential activation of p53-dependent G1 and G2 checkpoints in response to ribosomal stress

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Abstract The regulation of gene expression by non-coding RNAs has been indicated to play important roles in a wide array of biological processes, including control of cell differentiation, cell proliferation and stress responses. MicroRNA (miRNA), a member of small non-coding RNAs, has been defined as a regulator of gene expression at post-transcriptional levels. An important feature of miRNA is a multiple targeting of mRNAs leading to the efficient connection and disconnection of intracellular signaling networks with precise timing during animal development. Indeed, increasing bodies of evidence indicate that deregulation of miRNA disconnects intracellular networks that induce activation and inactivation of oncogenic and tumor-suppressive signaling pathways. We previously identified miR-101 as a candidate tumor-suppressor miRNA in colon cancer by the functional screening method. Here, we describe a novel function of miR-101 as a molecular switch for the surveillance system of DNA damage, which is established following activation of the p53-dependent checkpoint pathway in response to ribosomal stress, and also as a possible determinant to eliminate damaged cells. Methods: Cell proliferation and death assays were carried out after transient introduction of mature miR-101 by the lipofection method. mRNA expression profiles and network analysis in the presence or absence of miR-101 conditions were by an Agilent system. The cell sorter system was BD FACSArea II. Quantification of miRNA and mRNA was performed by qRT-PCR using TaqMan assay. Results: Introduction of miR-101 induced G2 arrest of the cell cycle and also apoptosis in p53 wild type cancer cell lines accompanied with accumulation of p53 stabilized by the elevation of phosphorylation at Ser15. mRNA expression profiles indicated that miR-101 induces an ATM-dependent DNA damage response pathway. To analyze this in detail, we screened potential miR-101 targets and identified several regulators of mitosis as direct targets, whose knock-down stabilizes p53 with elevated phosphorylation at Ser15. Furthermore, phosphorylation of p53 induced by miR-101 depended on the activation of ATM, without phosphorylation of H2AX, suggesting that miR-101 activates ATM without DNA-damage. Cell sorting analysis clearly indicated that phosphorylation of p53 occurs only in the G2 phase of the cell cycle, not in the G1 phase, and down-regulation of targets is also found in the G2-phase with increasing apoptotic cell death. These finding indicated the roles of miR-101, whose up-regulation activates the G2 specific checkpoint pathway, and blocks cell entry into mitosis. Furthermore, we found that ribosomal stress is an activator of miR-101 expression in a p53-dependent manner. In brief, when cells were exposed to CX-4561, a specific inhibitor of Pol I, p53 was immediately activated with phosphorylation of H2AX, indicating G1-S checkpoint for repair of DNA damage. Up-regulation of miR-101 was detected 36 hours after cells were exposed to ribosomal stress, in which ATM and p53 were continuously activated, and the majority of cells were arrested in the G2 phase of the cell cycle, accompanied with remarkable reduction of phosphorylation of H2AX and G2 specific miR-101 targets. These results suggest that miR-101 is associated with the establishment of the G2 checkpoint signaling pathway following the activation of the traditional p53 G1-S checkpoint, and responds to damaged cells through the blockage of cell entry into mitosis by repressing expression of target genes to prevent irregular cell division. Conclusion: Our results might indicate that miR-101 is required for the sequential activation of two different p53-dependent pathways in response to certain stresses, and dysfunction of miR-101 might confer a chance of survival for damaged cells with tumorigenic potential. Citation Format: Naoto Tsuchiya, Yuko Fujiwara, Hitoshi Nakagama, Takashi Kohno, Jun Yokota. MiR-101 as a molecular switch for sequential activation of p53-dependent G1 and G2 checkpoints in response to ribosomal stress. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A61.