The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.

The modulation of pre‐mRNA splicing is proposed as an attractive anti‐neoplastic strategy, especially for the cancers that exhibit aberrant pre‐mRNA splicing. Here, we discovered that T‐025 functions as an orally available and potent inhibitor of Cdc2‐like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T‐025 reduced CLK‐dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo. Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive‐associated biomarker of T‐025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T‐025 treatment. MYC activation, which altered pre‐mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti‐tumor efficacy of T‐025 in an allograft model of spontaneous, MYC‐driven breast cancer, at well‐tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC‐driven cancer patients.

In response to a shortage of intracellular energy, mammalian cells reduce energy consumption and induce cell cycle arrest, both of which contribute to cell survival. Here we report that a novel nucleolar pathway involving the energy-dependent nucleolar silencing complex (eNoSC) and Myb-binding protein 1a (MYBBP1A) is implicated in these processes. Namely, in response to glucose starvation, eNoSC suppresses rRNA transcription, which results in a reduction in nucleolar RNA content. As a consequence, MYBBP1A, which is anchored to the nucleolus via RNA, translocates from the nucleolus to the nucleoplasm. The translocated MYBBP1A induces acetylation and accumulation of p53 by enhancing the interaction between p300 and p53, which eventually leads to the cell cycle arrest (or apoptosis). Taken together, our results indicate that the nucleolus works as a sensor that transduces the intracellular energy status into the cell cycle machinery.Intracellular energy balance is important for cell survival. In response to nutrient or environmental stresses that cause reduction in the intracellular energy level, mammalian cells sense the intrinsic energy status and attempt to restore bioenergetic homeostasis through compensatory changes in the regulation of intermediate metabolic processes. One such mechanism is the reduction of ribosome biosynthesis, a major biosynthetic and energy-consuming process in mammalian cells, whereas the other mechanism is the arrest of cell proliferation. The LKB1-AMP-activated protein kinase (AMPK) 3 pathway is reportedly involved in both regulatory processes (1-5). During energy starvation or glucose deprivation, when the cellular AMP/ATP ratio is increased, the LKB1-AMPK pathway is activated. This signaling in turn inhibits the mammalian target of rapamycin (mTOR)/p70 S6 kinase activity that is required for rapid and sustained serum-induced ribosomal biosynthesis (2). In addition to this regulation, AMPK, which is reportedly activated in response to reduced energy level, phosphorylates and activates p53, which leads to G 1 cell cycle arrest (6). Inhibition of mTOR by AMPK and G 1 cell cycle arrest by AMPK via p53 activation suppresses energy expenditure and protects cells from energy deprivation-induced apoptosis (4, 5).On the other hand, a recent study in our laboratory revealed that glucose deprivation also reduces rRNA synthesis, which leads to down-regulation of ribosome biosynthesis in HeLa cells lacking the LKB1-AMPK pathway (7). We found that a novel nucleolar protein, nucleomethylin (NML), forms an energy-dependent nucleolar silencing complex (eNoSC) with SIRT1 and SUV39H1 and functions in this process. Our results suggest that an energy-dependent change in the NAD ϩ /NADH ratio regulates eNoSC, allowing the complex to couple the changing energy status with the level of rRNA transcription by regulating the epigenetic status of rRNA clusters. eNoSC promotes the restoration of energy balance and protects cells from energy deprivation-induced apoptosis (7).With regard to p53 activation, several...

The nucleolus, whose primary function is ribosome biogenesis, plays an essential role in p53 activation. Ribosome biogenesis is inhibited in response to cellular stress and several nucleolar proteins translocate from the nucleolus to the nucleoplasm, where they activate p53. In this study, we analysed precisely how impaired ribosome biogenesis regulates the activation of p53 by depleting nucleolar factors involved in rRNA transcription or rRNA processing. Nucleolar RNA content decreased when rRNA transcription was inhibited. In parallel with the reduced levels of nucleolar RNA content, the nucleolar protein Myb-binding protein 1 A (MYBBP1A) translocated to the nucleoplasm and increased p53 acetylation. The acetylated p53 enhanced p21 and BAX expression and induced apoptosis. In contrast, when rRNA processing was inhibited, MYBBP1A remained in the nucleolus and nonacetylated p53 accumulated, causing cell cycle arrest at the G1 phase by inducing p21 but not BAX. We propose that the nucleolus functions as a stress sensor to modulate p53 protein levels and its acetylation status, determining cell fate between cell cycle arrest and apoptosis by regulating MYBBP1A translocation.

Vacuolar (H+)‐ATPases (V‐ATPases) have important roles in the supply of nutrients to tumors by mediating autophagy and the endocytic uptake of extracellular fluids. Accordingly, V‐ATPases are attractive therapeutic targets for cancer. However, the clinical use of V‐ATPase inhibitors as anticancer drugs has not been realized, possibly owing to their high toxicity in humans. Inhibition of V‐ATPase may be an appropriate strategy in highly susceptible cancers. In this study, we explored markers of V‐ATPase inhibitor sensitivity. V‐ATPase inhibitors led to pH impairment in acidic intracellular compartments, suppression of macropinocytosis, and decreased intracellular amino acid levels. The sensitivity of cells to V‐ATPase inhibitors was correlated with low cathepsin D expression, and cancer cells showed increased sensitivity to V‐ATPase inhibitors after pretreatment with a cathepsin D inhibitor and siRNA targeting the cathepsin D gene (CTSD). In addition, V‐ATPase inhibitor treatment led to the induction of the amino acid starvation response, upregulation of endoplasmic reticulum stress markers, and suppression of mammalian target of rapamycin (mTOR) signaling in cells expressing low levels of cathepsin D. Some colorectal cancer patients showed the downregulation of cathepsin D in tumor tissues compared with matched normal tissues. These findings indicate that V‐ATPase inhibitors are promising therapeutic options for cancers with downregulated cathepsin D.