Cancer-associated MDM2 W329G mutant attenuates ribosomal stress-mediated p53 responses to promote cell survival and glycolysis.

Although amplification/overexpression is the predominant mechanism for the oncogenic properties of MDM2, an increasing number of MDM2 somatic missense mutations were identified in cancer patients with the recent advances in sequencing technology. Here, we characterized an MDM2 cancer-associated mutant variant W329G identified from a patient sample that contains a wild-type p53 gene. Trp329 is one of residues that were reported to be critical to MDM2's binding to ribosomal protein L11 (RPL11). We found that the MDM2 W329G mutant was resistant to the inhibitory effect of RPL11 on MDM2-mediated p53 ubiquitination and degradation, in line with its defect on RPL11 binding. Using isogenic U2OS cells with or without endogenous MDM2 W329G mutation, we demonstrated that the expression of classic p53 targets induced by ribosomal stress signals was reduced in mutant cells. RNA-seq analysis revealed that upon 5-FU treatment, the p53 response was significantly impaired. Also, the 5-FU-mediated repression of genes in cell cycle progression and DNA replication was diminished in W329G mutant-containing cells. Physiologically, U2OS W329G cells were more resistant to cell growth inhibition induced by ribosomal stress and exhibited higher glycolytic rates upon 5-FU treatment. Together, our data indicated that cancer-associated MDM2 W329G mutant attenuates ribosomal stress-mediated p53 responses to promote cell survival and glycolysis.

Emerging Role of GCN1 in Disease and Homeostasis.

GCN1 is recognized as a factor that is essential for the activation of GCN2, which is a sensor of amino acid starvation. This function is evolutionarily conserved from yeast to higher eukaryotes. However, recent studies have revealed non-canonical functions of GCN1 that are independent of GCN2, such as its participation in cell proliferation, apoptosis, and the immune response, beyond the borders of species. Although it is known that GCN1 and GCN2 interact with ribosomes to accomplish amino acid starvation sensing, recent studies have reported that GCN1 binds to disomes (i.e., ribosomes that collide each other), thereby regulating both the co-translational quality control and stress response. We propose that GCN1 regulates ribosome-mediated signaling by dynamically changing its partners among RWD domain-possessing proteins via unknown mechanisms. We recently demonstrated that GCN1 is essential for cell proliferation and whole-body energy regulation in mice. However, the manner in which ribosome-initiated signaling via GCN1 is related to various physiological functions warrants clarification. GCN1-mediated mechanisms and its interaction with other quality control and stress response signals should be important for proteostasis during aging and neurodegenerative diseases, and may be targeted for drug development.

Ribosomal Profiling by Gradient Fractionation of Cell Lysates.

Ribosomal profiling is a widely used technique for deep sequencing of ribosome-protected mRNA and for measuring ribosome status in cells. It is a powerful method that is typically employed for monitoring and measuring protein translation status and ribosome activity. Also, it has been used for monitoring the ribosomal stress-responsive events in the ribosome activity. Furthermore, this approach enables understanding of translational regulation, which is invisible in most proteomic approaches. Moreover, this method is known as an important approach for biological discovery such as identification of translation products. Hence, this methodology will be useful for studying cellular events engaging in ribosome assembly, ribosome biogenesis, ribosome activity, translation during the cell cycle, cell proliferation, and growth as well as the ribosomal stress response in mammalian cells.

FCN3 inhibits the progression of hepatocellular carcinoma by suppressing SBDS-mediated blockade of the p53 pathway.

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer deaths globally. Although considerable progress has been made in the treatment, clinical outcomes of HCC patients are still poor. Therefore, it is necessary to find novel prognostic factors upon which prevention and treatment strategies can be formulated. Ficolin-3 (FCN3) protein is a member of the human ficolin family. It activates complement through pathways associated with mannose-binding lectin-associated serine proteases. Herein, we identified that FCN3 was downregulated in HCC tissues and decreased FCN3 expression was closely related to poor prognosis. Overexpression of FCN3 induced apoptosis and inhibited cell proliferation via the p53 signaling pathway. Mechanistically, FCN3 modulated the nuclear translocation of eukaryotic initiation factor 6 (EIF6) by binding ribosome maturation factor (SBDS), which induced ribosomal stress and activation of the p53 pathway. In addition, Y-Box Binding Protein 1 (YBX1) involved in the transcription and translation level regulation of FCN3 to SBDS. Besides, a negative feedback loop in the downstream of FCN3 involving p53, YBX1 and SBDS was identified.

Xenobiotic-induced ribosomal stress compromises dysbiotic gut barrier aging: A one health perspective.

Upon exposure to internal or environmental insults, ribosomes stand sentinel. In particular, stress-driven dysregulation of ribosomal homeostasis is a potent trigger of adverse outcomes in mammalians. The present study assessed whether the ribosomal insult affects the aging process via the regulation of sentinel organs such as the gut. Analyses of the human aging dataset demonstrated that elevated features of ribosomal stress are inversely linked to barrier maintenance biomarkers during the aging process. Ribosome-insulted worms displayed reduced lifespan, which was associated with the disruption of gut barriers. Mechanistically, ribosomal stress-activated Sek-1/p38 signaling, a central platform of ribosomal stress responses, counteracted the gut barrier deterioration through the maintenance of the gut barrier, which was consistent with the results in a murine insult model. However, since the gut-protective p38 signaling was attenuated with aging, the ribosomal stress-induced distress was exacerbated in the gut epithelia and mucosa of the aged animals, subsequently leading to increased bacterial exposure. Moreover, the bacterial community-based evaluation predicted concomitant increases in the abundance of mucosal sugar utilizers and mucin metabolic enzymes in response to ribosomal insult in the aged host. All of the present evidence on ribosomal insulting against the gut barrier integrity from worms to mammals provides new insights into organelle-associated translational modulation of biological longevity in a one health perspective.

Missense mutation in RPS7 causes Diamond-Blackfan anemia via alteration of erythrocyte metabolism, protein translation and induction of ribosomal stress.

Diamond-Blackfan anemia (DBA) is predominantly underlined by mutations in genes encoding ribosomal proteins (RP); however, its etiology remains unexplained in approximately 25 % of patients. We previously reported a novel heterozygous RPS7 mutation hg38 chr2:g.3,580,153G > T p.V134F in one female patient and two asymptomatic family members, in whom mild anemia and increased erythrocyte adenosine deaminase (eADA) activity were detected. We observed that altered erythrocyte metabolism and oxidative stress which may negatively affect the lifespan of erythrocytes distinguishes the patient from her asymptomatic family members. Pathogenicity of the RPS7 p.V134F mutation was extensively validated including molecular defects in protein translational activity and ribosomal stress activation in the cellular model of this variant.

Olaparib Induces RPL5/RPL11-Dependent p53 Activation via Nucleolar Stress.

The poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) Olaparib is a widely used targeted therapy for a variety of solid tumors with homologous recombination deficiency (HRD) caused by mutation of BRCA1/2 or other DNA repair genes. The anti-tumor activity of Olaparib has been largely attributed to its ability to inhibit PARP enzymes and block DNA single-strand break (SSB) repair, which eventually leads to the most detrimental DNA damage, double-strand breaks (DSB), in HRD cells. Although PARPi was found to induce p53-dependent cell death, the underlying molecular mechanism remains incompletely understood. Here, we report that Olaparib treatment leads to p53 stabilization and activation of its downstream target genes in a dose- and time-dependent manner. Mechanistically, Olaparib triggers nucleolar stress by inhibiting biosynthesis of the precursor of ribosomal RNAs (pre-rRNA), resulting in enhanced interaction between ribosomal proteins (RPs), RPL5 and RPL11, and MDM2. Consistently, knockdown of RPL5 and RPL11 prevents Olaparib-induced p53 activation. More importantly, Olaparib efficiently suppresses breast and colorectal cancer cell survival and proliferation through activation of p53. Altogether, our study demonstrates that Olaparib activates the nucleolar stress-RPs-p53 pathway, suggesting rRNA biogenesis as a novel target for PARPi.

Inhibition of ribosomal RNA processing 15 Homolog (RRP15), which is overexpressed in hepatocellular carcinoma, suppresses tumour growth via induction of senescence and apoptosis.

Recent studies suggest that RRP15 (Ribosomal RNA Processing 15 Homolog) might be a potential target for cancer therapy. However, the role of RRP15 in hepatocarcinogenesis remains poorly delineated. In this study, we aimed to evaluate the expression and biological function of RRP15 in human hepatocellular carcinoma (HCC). We show that RRP15 was up regulated in HCC cell lines and tumours. Up-regulation of RRP15 in HCC tumours was also correlated with unfavorable prognosis. We further show that the frequent up-regulation of RRP15 in HCCs is at least partly driven by recurrent gene copy gain at chromosome 1q41. Functional studies indicated that RRP15 knockdown suppresses HCC proliferation and growth both in vitro and in vivo. Mechanistically, RRP15 depletion in p53-wild-type HepG2 cells induced senescence via activation of the p53-p21 signalling pathway through enhanced interaction of RPL11 with MDM2, as well as inhibition of SIRT1-mediated p53 deacetylation. Moreover, RRP15 depletion in p53-mutant PLC5 and p53-deleted Hep3B cells induced metabolic shift from the glycolytic pentose-phosphate to mitochondrial oxidative phosphorylation via regulating a series of key genes such as HK2 and TIGAR, and thus, promoted the generation of ROS and apoptosis. Taken together, our findings provide evidence for an important role of the RRP15 gene in hepatocarcinogenesis through regulation of HCC proliferation and growth, raising the possibility that targeting RRP15 may represent a potential therapeutic strategy for HCC treatment.

RPL32 Promotes Lung Cancer Progression by Facilitating p53 Degradation.

Lung cancer is the leading cause of cancer death worldwide, and the overall survival rate of advanced lung cancer patients is unsatisfactory. Ribosomal proteins (RPs) play important roles in carcinogenesis. However, the role of RPL32 in lung cancer has not been demonstrated. Here, we report that RPL32 is aberrantly, highly expressed in lung cancer tissues and that the overexpression of RPL32 is correlated with the poor prognosis of these patients. RPL32 silencing significantly inhibited the proliferation of lung cancer cells, with an observed p53 accumulation and cell-cycle arrest. Mechanistically, knockdown of RPL32 resulted in ribosomal stress and affected rRNA maturation. RPL5 and RPL11 sensed stress and translocated from the nucleus to the nucleoplasm, where they bound to murine double minute 2 (MDM2), an important p53 E3 ubiquitin ligase, which resulted in p53 accumulation and inhibition of cancer cell proliferation. As lung cancer cells usually express high levels of Toll-like receptor 9 (TLR9), we conjugated RPL32 small interfering RNA (siRNA) to the TLR9 ligand CpG to generate CpG-RPL32 siRNA, which could stabilize and guide RPL32 siRNA to lung cancer cells. Excitingly, CpG-RPL32 siRNA displayed strong anticancer abilities in lung cancer xenografts. Therefore, RPL32 is expected to be a potential target for lung cancer treatment.

Heavy ion radiation-induced DNA damage mediates apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway in mouse spermatogonia.

The risk of exposure to ionizing radiation (IR) environments has increased with the development of nuclear technology. IR exposure induces excessive apoptosis of the spermatogonia, which leads to male infertility. Spermatogonia apoptosis may be involved in ribosomal stress triggered by DNA damage following exposure to IR because ribosomal proteins (RPs) directly interact with mouse double minute 2 homolog (MDM2) to induce apoptosis. This study aimed to use comparative proteomics and transcriptomics approach to screen the differential RPs and ribosomal mRNAs in mouse testes following high linear energy transfer (LET) carbon ion radiation (CIR). The expression of ribosomal large subunit protein 27a (Rpl27a) decreased at both protein and mRNA levels in the spermatogonia in vivo. After 6 h of CIR, the immunofluorescence signal of 8-oxo-dG and phosphorylated ataxia-telangiectasia-mutated protein (ATM)/histone H2Ax increased, but that of Rpl27a decreased in the spermatogonia of p53 wild-type and knockout mouse testes. Moreover, the nucleolin was scattered throughout the nucleoplasm after CIR. These results suggested that CIR-induced DNA damage might trigger ribosomal stress, and the reduction in the expression of Rpl27a was associated with DNA damage in the spermatogonia. Similarly, in vitro, the immunofluorescence signal of 8-oxo-dG increased in the GC-1 cells after CIR. Moreover, the expression of Rpl27a was regulated by DNA damage because the co-transfection of ATM and Rpl27a or inhibition of ATM-treated CIR could restore the expression of Rpl27a. Furthermore, the reduction in the expression of Rpl27a led to weakened binding of E2F transcription factor 1 (E2F1) and p53 to MDM2, causing p53 activation and E2F1 degradation in p53 wild-type and knockdown GC-1 cells. This study proposed that heavy ion radiation-induced DNA damage mediated spermatogonia apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway. The results provided the underlying molecular mechanisms of spermatogonia apoptosis following exposure to high LET radiation.

USP47 Promotes Tumorigenesis by Negative Regulation of p53 through Deubiquitinating Ribosomal Protein S2.

p53 is activated in response to cellular stresses such as DNA damage, oxidative stress, and especially ribosomal stress. Although the regulations of p53 by E3 ligase and deubiquitinating enzymes (DUBs) have been described, the cellular roles of DUB associated with ribosomal stress have not been well studied. In this study, we report that Ubiquitin Specific Protease 47 (USP47) functions as an important regulator of p53. We show that ubiquitinated ribosomal protein S2 (RPS2) by Mouse double minute 2 homolog (MDM2) is deubiquitinated by USP47. USP47 inhibits the interaction between RPS2 and MDM2 thereby alleviating RPS2-mediated suppression of MDM2 under normal conditions. However, dissociation of USP47 leads to RPS2 binding to MDM2, which is required for the suppression of MDM2, consequently inducing up-regulation of the p53 level under ribosomal stress. Finally, we show that depletion of USP47 induces p53 and therefore inhibits cell proliferation, colony formation, and tumor progression in cancer cell lines and a mouse xenograft model. These findings suggest that USP47 could be a potential therapeutic target for cancer.

Ribosomal/nucleolar stress induction regulates tert-Butyl hydroperoxide (tBHP) mediated oxidative stress in Anopheles gambiae midguts.

OBJECTIVE: A fundamental understanding of redox homeostasis in Anopheles gambiae midgut cells under different oxidative conditions is missing. Such knowledge can aid in the development of new malaria transmission-blocking strategies aimed at disrupting natural homeostatic processes in the mosquito during Plasmodium parasite uptake (i.e. blood feeding). The aim of this study was to understand how the An. gambiae midgut regulates oxidative stress to reactive oxygen species (ROS), especially to a potent ROS-inducer such as tert-Butyl hydroperoxide (tBHP). RESULTS: Initial studies using quantitative immunoblot indicated that the expression of the classical antioxidant protein An. gambiae thioredoxin-1 (AgTrx-1) remained unchanged across challenges with different concentrations of tBHP suggesting that additional mechanisms to regulate ROS may be involved. We therefore conducted a global proteomic survey, which revealed that An. gambiae midguts under low (50 µM) and high (200 µM) tBHP concentrations were enriched in proteins indicative of ribosomal/nucleolar stress. Ribosomal stress is an inherent cellular response to an imbalance in ribosomal proteins (RPs) due to cellular stress such as oxidative stress. Our data suggest that ribosomal/nucleolar stress is the primary cellular response in An. gambiae midguts under tBHP challenge. Considering these results, we discuss harnessing the ribosomal stress response as a potential malaria transmission-blocking strategy.

Pre-Ribosomal RNA Processing in Human Cells: From Mechanisms to Congenital Diseases.

Ribosomal RNAs, the most abundant cellular RNA species, have evolved as the structural scaffold and the catalytic center of protein synthesis in every living organism. In eukaryotes, they are produced from a long primary transcript through an intricate sequence of processing steps that include RNA cleavage and folding and nucleotide modification. The mechanisms underlying this process in human cells have long been investigated, but technological advances have accelerated their study in the past decade. In addition, the association of congenital diseases to defects in ribosome synthesis has highlighted the central place of ribosomal RNA maturation in cell physiology regulation and broadened the interest in these mechanisms. Here, we give an overview of the current knowledge of pre-ribosomal RNA processing in human cells in light of recent progress and discuss how dysfunction of this pathway may contribute to the physiopathology of congenital diseases.

Proteomic profiling reveals DNA damage, nucleolar and ribosomal stress are the main responses to oxaliplatin treatment in cancer cells.

Oxaliplatin is widely used to treat colorectal cancer in both palliative and adjuvant settings. It is also being tested for use in treating hematological, esophageal, biliary tract, pancreatic, gastric, and hepatocellular cancers. Despite its routine clinical use, little is known about the responses it induces in cancer cells. Therefore the whole-cell proteomics study was conducted to characterize the cellular response induced by oxaliplatin. Chemosensitive CCRF-CEM cells were treated with oxaliplatin at 29.3µM (5×IC50) for 240min (half-time to caspase activation). The proteomes of un-/treated cells were then compared by high-resolution mass spectrometry, revealing 4049 proteins expressed over 3 biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in at least two replicates. In agreement with the DNA-damaging effects of platinum drugs, proteins involved in DNA damage responses were present in both the upregulated and downregulated groups. The downregulated proteins were divided into three subgroups; i) centrosomal proteins, ii) RNA processing and iii) ribosomal proteins, which indicates nucleolar and ribosomal stress. In conclusion, our data supported by further validation experiments indicate the initial cellular response to oxaliplatin is the activation of DNA damage response, which in turn or in parallel triggers nucleolar and ribosomal stress. BIOLOGICAL SIGNIFICANCE: We have performed a whole-cell proteomic study of cellular response to oxaliplatin treatment, which is the drug predominantly used in the treatment of colorectal cancer. Compared to its predecessors, cisplatin and carboplatin, there is only a small fraction of studies dedicated to oxaliplatin. From those studies, most of them are focused on modification of treatment regimens or study of oxaliplatin in new cancer diagnoses. Cellular response hasn't been studied deeply and to our best knowledge, this is the first whole-cell proteomics study focused exclusively to this important topic, which can help to understand molecular mechanisms of action.

BCCIPß modulates the ribosomal and extraribosomal function of S7 through a direct interaction.

Extraribosomal functions of ribosomal proteins (RPs) have gained much attention for their implications in tumorigenesis and progression. However, the regulations for transition between the ribosomal and extraribosomal functions of RPs are rarely reported. Herein, we identified a ribosomal protein S7-interacting partner, BCCIPß, which modulates the functional conversion of S7. Through the N-terminal acidic domain, BCCIPß interacts with the central basic region in S7 and regulates the extraribosomal distribution of S7. BCCIPß deficiency abrogates the ribosomal accumulation but enhances the ribosome-free location of S7. This translocation further impairs protein synthesis and triggers ribosomal stress. Consequently, BCCIPß deficiency suppresses the ribosomal function and initiates the extraribosomal function of S7, resulting in restriction of cell proliferation. Moreover, clinically relevant S7 mutations were found to dampen the interaction with BCCIPß and facilitate the functional transition of S7. In conclusion, BCCIPß, as a S7 modulator, contributes to the regulation of ribosomal and extraribosomal functions of S7 and has implications in cell growth and tumor development.

Acrolein preferentially damages nucleolus eliciting ribosomal stress and apoptosis in human cancer cells.

Acrolein (Acr) is a potent cytotoxic and DNA damaging agent which is ubiquitous in the environment and abundant in tobacco smoke. Acr is also an active cytotoxic metabolite of the anti-cancer drugs cyclophosphamide and ifosfamide. The mechanisms via which Acr exerts its anti-cancer activity and cytotoxicity are not clear. In this study, we found that Acr induces cytotoxicity and cell death in human cancer cells with different activities of p53. Acr preferentially binds nucleolar ribosomal DNA (rDNA) to form Acr-deoxyguanosine adducts, and induces oxidative damage to both rDNA and ribosomal RNA (rRNA). Acr triggers ribosomal stress responses, inhibits rRNA synthesis, reduces RNA polymerase I binding to the promoter of rRNA gene, disrupts nucleolar integrity, and impairs ribosome biogenesis and polysome formation. Acr causes an increase in MDM2 levels and phosphorylation of MDM2 in A549 and HeLa cells which are p53 active and p53 inactive, respectively. It enhances the binding of ribosomal protein RPL11 to MDM2 and reduces the binding of p53 and E2F-1 to MDM2 resulting in stabilization/activation of p53 in A549 cells and degradation of E2F-1 in A549 and HeLa cells. We propose that Acr induces ribosomal stress which leads to activation of MDM2 and RPL11-MDM2 binding, consequently, activates p53 and enhances E2F-1 degradation, and that taken together these two processes induce apoptosis and cell death.

The role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity.

Ribosomal proteins (RPs), the essential components of the ribosome, are a family of RNA-binding proteins, which play prime roles in ribosome biogenesis and protein translation. Recent studies revealed that RPs have additional extra-ribosomal functions, independent of protein biosynthesis, in regulation of diverse cellular processes. Here, we review recent advances in our understanding of how RPs regulate apoptosis, cell cycle arrest, cell proliferation, neoplastic transformation, cell migration and invasion, and tumorigenesis through both MDM2/p53-dependent and p53-independent mechanisms. We also discuss the roles of RPs in the maintenance of genome integrity via modulating DNA damage response and repair. We further discuss mutations or deletions at the somatic or germline levels of some RPs in human cancers as well as in patients of Diamond-Blackfan anemia and 5q- syndrome with high susceptibility to cancer development. Moreover, we discuss the potential clinical application, based upon abnormal levels of RPs, in biomarker development for early diagnosis and/or prognosis of certain human cancers. Finally, we discuss the pressing issues in the field as future perspectives for better understanding the roles of RPs in human cancers to eventually benefit human health.

Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development.

HEY1 functions are regulated by its phosphorylation at Ser-68.

HEY1 (hairy/enhancer-of-split related with YRPW motif 1) is a member of the basic helix-loop-helix-orange (bHLH-O) family of transcription repressors that mediate Notch signalling. HEY1 acts as a positive regulator of the tumour suppressor p53 via still unknown mechanisms. A MALDI-TOF/TOF MS analysis has uncovered a novel HEY1 regulatory phosphorylation event at Ser-68. Strikingly, this single phosphorylation event controls HEY1 stability and function: simulation of HEY1 Ser-68 phosphorylation increases HEY1 protein stability but inhibits its ability to enhance p53 transcriptional activity. Unlike wild-type HEY1, expression of the phosphomimetic mutant HEY1-S68D failed to induce p53-dependent cell cycle arrest and it did not sensitize U2OS cells to p53-activating chemotherapeutic drugs. We have identified two related kinases, STK38 (serine/threonine kinase 38) and STK38L (serine/threonine kinase 38 like), which interact with and phosphorylate HEY1 at Ser-68. HEY1 is phosphorylated at Ser-68 during mitosis and it accumulates in the centrosomes of mitotic cells, suggesting a possible integration of HEY1-dependent signalling in centrosome function. Moreover, HEY1 interacts with a subset of p53-activating ribosomal proteins. Ribosomal stress causes HEY1 relocalization from the nucleoplasm to perinucleolar structures termed nucleolar caps. HEY1 interacts physically with at least one of the ribosomal proteins, RPL11, and both proteins cooperate in the inhibition of MDM2-mediated p53 degradation resulting in a synergistic positive effect on p53 transcriptional activity. HEY1 itself also interacts directly with MDM2 and it is subjected to MDM2-mediated degradation. Simulation of HEY1 Ser-68 phosphorylation prevents its interaction with p53, RPL11 and MDM2 and abolishes HEY1 migration to nucleolar caps upon ribosomal stress. Our findings uncover a novel mechanism for cross-talk between Notch signalling and nucleolar stress.

PIM1 destabilization activates a p53-dependent response to ribosomal stress in cancer cells.

Defects in ribosome biogenesis triggers a stress response (ribosomal stress) that can lead to growth arrest and apoptosis. Signaling pathways activated by ribosomal stress are specifically involved in the pathological mechanism of a group of disorders defined as ribosomopathies. However, more generally, the quality control of ribosome synthesis is part of the regulatory circuits that control cell metabolism. A number of studies identified tumor suppressor p53 as a central player in ribosomal stress. We have previously reported that the kinase PIM1 plays a role as a sensor for ribosome deficiency. In this report we address the relationship between PIM1 and p53 in cancer cell lines after depletion of a ribosomal protein. We identified a novel signaling pathway that includes the kinase AKT and the ubiquitin ligase MDM2. In fact, our results indicate that the lower level of PIM1, induced by ribosomal stress, causes inactivation of AKT, inhibition of MDM2 and a consequent p53 stabilization. Therefore, we propose that activation of p53 in response to ribosomal stress, is dependent on the pathway PIM1-AKT-MDM2. In addition, we report evidence that PIM1 level may be relevant to assess the sensitivity of cancer cells to chemotherapeutic drugs that induce ribosomal stress.