Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism.

Dis3L2 is a highly conserved 3'-5' exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms' tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.

Probing the Anticancer Action of Novel Ferrocene Analogues of MNK Inhibitors.

Two novel ferrocene-containing compounds based upon a known MNK1/2 kinase (MAPK-interacting kinase) inhibitor have been synthesized. The compounds were designed to use the unique shape of ferrocene to exploit a large hydrophobic pocket in MNK1/2 that is only partially occupied by the original compound. Screening of the ferrocene analogues showed that both exhibited potent anticancer effects in several breast cancer and AML (acute myeloid leukemia) cell lines, despite a loss of MNK potency. The most potent ferrocene-based compound 5 was further analysed in vitro in MDA-MB-231 (triple negative breast cancer cells). Dose⁻response curves of compound 5 for 2D assay and 3D assay generated IC50 values (half maximal inhibitory concentration) of 0.55 µM and 1.25 µM, respectively.

Synergistic effects of inhibiting the MNK-eIF4E and PI3K/AKT/ mTOR pathways on cell migration in MDA-MB-231 cells.

The study of eukaryotic initiation factor 4E (eIF4E) is a key focus in cancer research due to its role in controlling the translation of tumour-associated proteins, that drive an aggressive migratory phenotype. eIF4E is a limiting component of the eIF4F complex which is a critical determinant for the translation of mRNAs. Mitogen-activated protein kinase interacting protein kinases (MNK1/2) phosphorylate eIF4E on Ser209, promoting the expression of oncogenic proteins, whereas mTORC1 phosphorylates and de-activates the eIF4E inhibitor, 4E-BP1, to release translational repression. Here we show that inhibiting these pathways simultaneously effectively slows the rate of cell migration in breast cancer cells. However, a molecular hybridisation approach using novel, cleavable dual MNK1/2 and PI3K/mTOR inhibiting hybrid agents was less effective at slowing cell migration.

Dual abrogation of MNK and mTOR: a novel therapeutic approach for the treatment of aggressive cancers.

Targeting the translational machinery has emerged as a promising therapeutic option for cancer treatment. Cancer cells require elevated protein synthesis and exhibit augmented activity to meet the increased metabolic demand. Eukaryotic translation initiation factor 4E is necessary for mRNA translation, its availability and phosphorylation are regulated by the PI3K/AKT/mTOR and MNK1/2 pathways. The phosphorylated form of eIF4E drives the expression of oncogenic proteins including those involved in metastasis. In this article, we will review the role of eIF4E in cancer, its regulation and discuss the benefit of dual inhibition of upstream pathways. The discernible interplay between the MNK and mTOR signaling pathways provides a novel therapeutic opportunity to target aggressive migratory cancers through the development of hybrid molecules.

The helicase, DDX3X, interacts with poly(A)-binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading.

DDX3X, a helicase, can interact directly with mRNA and translation initiation factors, regulating the selective translation of mRNAs that contain a structured 5' untranslated region. This activity modulates the expression of mRNAs controlling cell cycle progression and mRNAs regulating actin dynamics, contributing to cell adhesion and motility. Previously, we have shown that ribosomes and translation initiation factors localise to the leading edge of migrating fibroblasts in loci enriched with actively translating ribosomes, thereby promoting steady-state levels of ArpC2 and Rac1 proteins at the leading edge of cells during spreading. As DDX3X can regulate Rac1 levels, cell motility and metastasis, we have examined DDX3X protein interactions and localisation using many complementary approaches. We now show that DDX3X can physically interact and co-localise with poly(A)-binding protein 1 and caprin-1 at the leading edge of spreading cells. Furthermore, as depletion of DDX3X leads to decreased cell motility, this provides a functional link between DDX3X, caprin-1 and initiation factors at the leading edge of migrating cells to promote cell migration and spreading.

Sumoylation of eIF4A2 affects stress granule formation.

Regulation of protein synthesis is crucial for cells to maintain viability and to prevent unscheduled proliferation that could lead to tumorigenesis. Exposure to stress results in stalling of translation, with many translation initiation factors, ribosomal subunits and mRNAs being sequestered into stress granules or P bodies. This allows the re-programming of the translation machinery. Many aspects of translation are regulated by post-translational modification. Several proteomic screens have identified translation initiation factors as targets for sumoylation, although in many cases the role of this modification has not been determined. We show here that eIF4A2 is modified by SUMO, with sumoylation occurring on a single residue (K226). We demonstrate that sumoylation of eIF4A2 is modestly increased in response to arsenite and ionising radiation, but decreases in response to heat shock or hippuristanol. In arsenite-treated cells, but not in hippuristanol-treated cells, eIF4A2 is recruited to stress granules, suggesting sumoylation of eIF4A2 correlates with its recruitment to stress granules. Furthermore, we demonstrate that the inability to sumoylate eIF4A2 results in impaired stress granule formation, indicating a new role for sumoylation in the stress response.

Murine norovirus 1 (MNV1) replication induces translational control of the host by regulating eIF4E activity during infection.

Protein synthesis is a tightly controlled process responding to several stimuli, including viral infection. As obligate intracellular parasites, viruses depend on the translation machinery of the host and can manipulate it by affecting the availability and function of specific eukaryotic initiation factors (eIFs). Human norovirus is a member of the Caliciviridae family and is responsible for gastroenteritis outbreaks. Previous studies on feline calicivirus and murine norovirus 1 (MNV1) demonstrated that the viral protein, genome-linked (VPg), acts to direct translation by hijacking the host protein synthesis machinery. Here we report that MNV1 infection modulates the MAPK pathway to activate eIF4E phosphorylation. Our results show that the activation of p38 and Mnk during MNV1 infection is important for MNV1 replication. Furthermore, phosphorylated eIF4E relocates to the polysomes, and this contributes to changes in the translational state of specific host mRNAs. We propose that global translational control of the host by eIF4E phosphorylation is a key component of the host-pathogen interaction.

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation.

Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for myogenic differentiation.

mTOR inhibition and levels of the DNA repair protein MGMT in T98G glioblastoma cells.

BACKGROUND: Glioblastoma multiforme (GBM), the most common and most aggressive type of primary adult brain tumour, responds poorly to conventional treatment. Temozolomide (TMZ) chemotherapy remains the most commonly used treatment, despite a large proportion of tumours displaying TMZ resistance. 60% of GBM tumours have unmethylated MGMT promoter regions, resulting in an overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is responsible for tumour resistance to TMZ chemotherapy. Tumours also often exhibit hyperactive PI3-kinase/mTOR signalling, which enables them to resynthesise proteins quickly. Since MGMT is a suicide protein that is degraded upon binding to and repairing TMZ-induced O6-methylguanine adducts, it has been hypothesized that inhibition of translation via the mTOR signalling pathway could generate a tumour-specific reduction in MGMT protein and increase TMZ sensitivity. METHODS: MGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro. RESULTS: We show that inhibiting mTOR signalling is indeed associated with acute inhibition of protein synthesis. Western blots show that despite this, relative to loading control proteins, steady state levels of MGMT protein increased and MGMT mRNA was retained in heavy polysomes. Whilst TMZ treatment resulted in maintained MGMT protein levels, concomitant treatment of T98G cells with TMZ and KU0063794 resulted in increased MGMT protein levels without changes in total mRNA levels. CONCLUSIONS: These in vitro data suggest that, counterintuitively, mTOR inhibition may not be a useful adjunct to TMZ therapy and that more investigation is needed before applying mTOR inhibitors in a clinical setting.

The S. pombe translation initiation factor eIF4G is Sumoylated and associates with the SUMO protease Ulp2.

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering protein-protein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively.

Weighing up the possibilities: Controlling translation by ubiquitylation and sumoylation.

Regulation of protein synthesis is of fundamental importance to cells. It has a critical role in the control of gene expression, and consequently cell growth and proliferation. The importance of this control is supported by the fact that protein synthesis is frequently upregulated in tumor cells. The major point at which regulation occurs is the initiation stage. Initiation of translation involves the interaction of several proteins to form the eIF4F complex, the recognition of the mRNA by this complex, and the subsequent recruitment of the 40S ribosomal subunit to the mRNA. This results in the formation of the 48S complex that then scans the mRNA for the start codon, engages the methionyl-tRNA and eventually forms the mature 80S ribosome which is elongation-competent. Formation of the 48S complex is regulated by the availability of individual initiation factors and through specific protein-protein interactions. Both of these events can be regulated by post-translational modification by ubiquitin or Ubls (ubiquitin-like modifiers) such as SUMO or ISG15. We provide here a summary of translation initiation factors that are modified by ubiquitin or Ubls and, where they have been studied in detail, describe the role of these modifications and their effects on regulating protein synthesis.

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis.

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

mRNA encoding WAVE-Arp2/3-associated proteins is co-localized with foci of active protein synthesis at the leading edge of MRC5 fibroblasts during cell migration.

During cell spreading, mammalian cells migrate using lamellipodia formed from a large dense branched actin network which produces the protrusive force required for leading edge advancement. The formation of lamellipodia is a dynamic process and is dependent on a variety of protein cofactors that mediate their local regulation, structural characteristics and dynamics. In the present study, we show that mRNAs encoding some structural and regulatory components of the WAVE [WASP (Wiskott-Aldrich syndrome protein) verprolin homologous] complex are localized to the leading edge of the cell and associated with sites of active translation. Furthermore, we demonstrate that steady-state levels of ArpC2 and Rac1 proteins increase at the leading edge during cell spreading, suggesting that localized protein synthesis has a pivotal role in controlling cell spreading and migration.

Multiple isoforms of the translation initiation factor eIF4GII are generated via use of alternative promoters, splice sites and a non-canonical initiation codon.

During the initiation stage of eukaryotic mRNA translation, the eIF4G (eukaryotic initiation factor 4G) proteins act as an aggregation point for recruiting the small ribosomal subunit to an mRNA. We previously used RNAi (RNA interference) to reduce expression of endogenous eIF4GI proteins, resulting in reduced protein synthesis rates and alterations in the morphology of cells. Expression of EIF4G1 cDNAs, encoding different isoforms (f-a) which arise through selection of alternative initiation codons, rescued translation to different extents. Furthermore, overexpression of the eIF4GII paralogue in the eIF4GI-knockdown background was unable to restore translation to the same extent as eIF4GIf/e isoforms, suggesting that translation events governed by this protein are different. In the present study we show that multiple isoforms of eIF4GII exist in mammalian cells, arising from multiple promoters and alternative splicing events, and have identified a non-canonical CUG initiation codon which extends the eIF4GII N-terminus. We further show that the rescue of translation in eIF4GI/eIF4GII double-knockdown cells by our novel isoforms of eIF4GII is as robust as that observed with either eIF4GIf or eIF4GIe, and more than that observed with the original eIF4GII. As the novel eIF4GII sequence diverges from eIF4GI, these data suggest that the eIF4GII N-terminus plays an alternative role in initiation factor assembly.

SMG-1 and mTORC1 act antagonistically to regulate response to injury and growth in planarians.

Planarian flatworms are able to both regenerate their whole bodies and continuously adapt their size to nutrient status. Tight control of stem cell proliferation and differentiation during these processes is the key feature of planarian biology. Here we show that the planarian homolog of the phosphoinositide 3-kinase-related kinase (PIKK) family member SMG-1 and mTOR complex 1 components are required for this tight control. Loss of smg-1 results in a hyper-responsiveness to injury and growth and the formation of regenerative blastemas that remain undifferentiated and that lead to lethal ectopic outgrowths. Invasive stem cell hyper-proliferation, hyperplasia, hypertrophy, and differentiation defects are hallmarks of this uncontrolled growth. These data imply a previously unappreciated and novel physiological function for this PIKK family member. In contrast we found that planarian members of the mTOR complex 1, tor and raptor, are required for the initial response to injury and blastema formation. Double smg-1 RNAi experiments with tor or raptor show that abnormal growth requires mTOR signalling. We also found that the macrolide rapamycin, a natural compound inhibitor of mTORC1, is able to increase the survival rate of smg-1 RNAi animals by decreasing cell proliferation. Our findings support a model where Smg-1 acts as a novel regulator of both the response to injury and growth control mechanisms. Our data suggest the possibility that this may be by suppressing mTOR signalling. Characterisation of both the planarian mTORC1 signalling components and another PIKK family member as key regulators of regeneration and growth will influence future work on regeneration, growth control, and the development of anti-cancer therapies that target mTOR signalling.

Ischemia-induced calpain activation causes eukaryotic (translation) initiation factor 4G1 (eIF4GI) degradation, protein synthesis inhibition, and neuronal death.

Persistent protein synthesis inhibition (PSI) is a robust predictor of eventual neuronal death following cerebral ischemia. We thus tested the hypothesis that persistent PSI inhibition and neuronal death are causally linked. Neuronal viability strongly correlated with both protein synthesis and levels of eukaryotic (translation) initiation factor 4G1 (eIF4G1). We determined that in vitro ischemia activated calpain, which degraded eIF4G1. Overexpression of the calpain inhibitor calpastatin or eIF4G1 resulted in increased protein synthesis and increased neuronal viability compared with controls. The neuroprotective effect of eIF4G1 overexpression was due to restoration of cap-dependent protein synthesis, as well as protein synthesis-independent mechanisms, as inhibition of protein synthesis with cycloheximide did not completely prevent the protective effect of eIF4G1 overexpression. In contrast, shRNA-mediated silencing of eIF4G1 exacerbated ischemia-induced neuronal injury, suggesting eIF4G1 is necessary for maintenance of neuronal viability. Finally, calpain inhibition following global ischemia in vivo blocked decreases in eIF4G1, facilitated protein synthesis, and increased neuronal viability in ischemia-vulnerable hippocampal CA1 neurons. Collectively, these data demonstrate that calpain-mediated degradation of a translation initiation factor, eIF4G1, is a cause of both persistent PSI and neuronal death.

Translation initiation factors and active sites of protein synthesis co-localize at the leading edge of migrating fibroblasts.

Cell migration is a highly controlled essential cellular process, often dysregulated in tumour cells, dynamically controlled by the architecture of the cell. Studies involving cellular fractionation and microarray profiling have previously identified functionally distinct mRNA populations specific to cellular organelles and architectural compartments. However, the interaction between the translational machinery itself and cellular structures is relatively unexplored. To help understand the role for the compartmentalization and localized protein synthesis in cell migration, we have used scanning confocal microscopy, immunofluorescence and a novel ribopuromycylation method to visualize translating ribosomes. In the present study we show that eIFs (eukaryotic initiation factors) localize to the leading edge of migrating MRC5 fibroblasts in a process dependent on TGN (trans-Golgi network) to plasma membrane vesicle transport. We show that eIF4E and eIF4GI are associated with the Golgi apparatus and membrane microdomains, and that a proportion of these proteins co-localize to sites of active translation at the leading edge of migrating cells.

Mitotic DNA damage targets the Aurora A/TPX2 complex.

We have previously shown that the DNA damage-induced G2 arrest is contributed by inhibition of Aurora A (AurA) and that transduction of active AurA into arrested cells allows bypassing the block through reactivation of CDK1. In this study, we investigated the mechanism of DNA damage-induced AurA inhibition. We provide evidence that ionizing radiation (IR) administered in mitosis, a time when AurA protein and enzymatic activity reach peak levels, impairs interaction with the partner TPX2, leading to inactivation of the kinase through dephosphorylation of AurA T-loop residue, T288. We find that decreased AurA-TPX2 complex formation in response to irradiation results from reduced cellular levels of TPX2, an effect that is both contributed by increased APC/CDH1-dependent protein degradation and decreased translation of TPX2 mRNA.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing.

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m(7)GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m(7)GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m(7)GTP-5(')cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex.