Autogenous Control of 5'TOP mRNA Stability by 40S Ribosomes.

Ribosomal protein (RP) expression in higher eukaryotes is regulated translationally through the 5'TOP sequence. This mechanism evolved to more rapidly produce RPs on demand in different tissues. Here we show that 40S ribosomes, in a complex with the mRNA binding protein LARP1, selectively stabilize 5'TOP mRNAs, with disruption of this complex leading to induction of the impaired ribosome biogenesis checkpoint (IRBC) and p53 stabilization. The importance of this mechanism is underscored in 5q− syndrome, a macrocytic anemia caused by a large monoallelic deletion, which we found to also encompass the LARP1 gene. Critically, depletion of LARP1 alone in human adult CD34+ bone marrow precursor cells leads to a reduction in 5'TOP mRNAs and the induction of p53. These studies identify a 40S ribosome function independent of those in translation that, with LARP1, mediates the autogenous control of 5'TOP mRNA stability, whose disruption is implicated in the pathophysiology of 5q− syndrome.

Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability.

Corrigendum: mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

This corrects the article DOI: 10.1038/nature22964.

Impaired ribosome biogenesis checkpoint activation induces p53-dependent MCL-1 degradation and MYC-driven lymphoma death.

MYC-driven B-cell lymphomas are addicted to increased levels of ribosome biogenesis (RiBi), offering the potential for therapeutic intervention. However, it is unclear whether inhibition of RiBi suppresses lymphomagenesis by decreasing translational capacity and/or by p53 activation mediated by the impaired RiBi checkpoint (IRBC). Here we generated Eµ-Myc lymphoma cells expressing inducible short hairpin RNAs to either ribosomal protein L7a (RPL7a) or RPL11, the latter an essential component of the IRBC. The loss of either protein reduced RiBi, protein synthesis, and cell proliferation to similar extents. However, only RPL7a depletion induced p53-mediated apoptosis through the selective proteasomal degradation of antiapoptotic MCL-1, indicating the critical role of the IRBC in this mechanism. Strikingly, low concentrations of the US Food and Drug Administration-approved anticancer RNA polymerase I inhibitor Actinomycin D (ActD) dramatically prolonged the survival of mice harboring Trp53+/+;Eµ-Myc but not Trp53-/-;Eµ-Myc lymphomas, which provides a rationale for treating MYC-driven B-cell lymphomas with ActD. Importantly, the molecular effects of ActD on Eµ-Myc cells were recapitulated in human B-cell lymphoma cell lines, highlighting the potential for ActD as a therapeutic avenue for p53 wild-type lymphoma.

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

A liaison between mTOR signaling, ribosome biogenesis and cancer.

The ability to translate genetic information into functional proteins is considered a landmark in evolution. Ribosomes have evolved to take on this responsibility and, although there are some differences in their molecular make-up, both prokaryotes and eukaryotes share a common structural architecture and similar underlying mechanisms of protein synthesis. Understanding ribosome function and biogenesis has been the focus of extensive research since the early days of their discovery. In the last decade however, new and unexpected roles have emerged that place deregulated ribosome biogenesis and protein synthesis at the crossroads of pathological settings, particularly cancer, revealing a set of novel cellular checkpoints. Moreover, it is also becoming evident that mTOR signaling, which regulates an array of anabolic processes, including ribosome biogenesis, is often exploited by cancer cells to sustain proliferation through the upregulation of global protein synthesis. The use of pharmacological agents that interfere with ribosome biogenesis and mTOR signaling has proven to be an effective strategy to control cancer development clinically. Here we discuss the most recent findings concerning the underlying mechanisms by which mTOR signaling controls ribosome production and the potential impact of ribosome biogenesis in tumor development. This article is part of a Special Issue entitled: Translation and Cancer.

BAG3 expression in glioblastoma cells promotes accumulation of ubiquitinated clients in an Hsp70-dependent manner.

Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners.

Induction of Id-1 by FGF-2 involves activity of EGR-1 and sensitizes neuroblastoma cells to cell death.

Inhibitor of differentiation-1 (Id-1) is a member of helix-loop-helix (HLH) family of proteins that regulate gene transcription through their inhibitory binding to basic-HLH transcription factors. Similarly to other members of this family, Id-1 is involved in the repression of cell differentiation and activation of cell growth. The dual function of Id-1, inhibition of differentiation, and stimulation of cell proliferation, might be interdependent, as cell differentiation is generally coupled with the exit from the cell cycle. Fibroblast growth factor-2 (FGF-2) has been reported to play multiple roles in different biological processes during development of the central nervous system (CNS). In addition, FGF-2 has been described to induce "neuronal-like" differentiation and trigger apoptosis in neuroblastoma SK-N-MC cells. Although regulation of Id-1 protein by several mitogenic factors is well-established, little is known about the role of FGF-2 in the regulation of Id-1. Using human neuroblastoma cell line, SK-N-MC, we found that treatment of these cells with FGF-2 resulted in early induction of both Id-1 mRNA and protein. The induction occurs within 1 h from FGF-2 treatment and is mediated by ERK1/2 pathway, which in turn stimulates expression of the early growth response-1 (Egr-1) transcription factor. We also demonstrate direct interaction of Egr-1 with Id-1 promoter in vitro and in cell culture. Finally, inhibition of Id-1 expression results in G(2) /M accumulation of FGF-2-treated cells and delayed cell death.

The 40S-LARP1 complex reprograms the cellular translatome upon mTOR inhibition to preserve the protein synthetic capacity.

Ribosomes execute the transcriptional program in every cell. Critical to sustain nearly all cellular activities, ribosome biogenesis requires the translation of ~200 factors of which 80 are ribosomal proteins (RPs). As ribosome synthesis depends on RP mRNA translation, a priority within the translatome architecture should exist to ensure the preservation of ribosome biogenesis capacity, particularly under adverse growth conditions. Here, we show that under critical metabolic constraints characterized by mTOR inhibition, LARP1 complexed with the 40S subunit protects from ribophagy the mRNAs regulon for ribosome biogenesis and protein synthesis, acutely preparing the translatome to promptly resume ribosomes production after growth conditions return permissive. Characterizing the LARP1-protected translatome revealed a set of 5'TOP transcript isoforms other than RPs involved in energy production and in mitochondrial function, among other processes, indicating that the mTOR-LARP1-5'TOP axis acts at the translational level as a primary guardian of the cellular anabolic capacity.

Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis.

The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis.

Autoregulation of co-chaperone BAG3 gene transcription.

The Bcl-2-associated athanogene, BAG, protein family through their BAG domain associates with the heat shock protein 70 (HSP-70) and modulates its chaperone activity. One member of this family, BAG3, appears to play an important role in protein homeostasis, as its expression promotes cell survival. Expression of BAG3 is enhanced by a variety of stress-inducing agents. Here we describe a role for BAG3 to modulate transcription of its own promoter through a positive feedback loop involving its 5'-UTR sequence. Activation of the BAG3 promoter is mediated by the BAG domain and is independent of BAG3 association with the UTR sequence. Autoactivation of the BAG3 gene is observed in several cultures of human glial cells including gliomas, but not in several other non-glial cell lines such as He La and others. Results from cell fractionation and immunocytochemistry showed BAG3 in the cytoplasm as well as the nuclei of glial cells. These observations suggest that BAG3 gene expression is controlled by its own product and that this may be critical for the biological activity of BAG3 in some cell types.

Detection of JC virus DNA fragments but not proteins in normal brain tissue.

OBJECTIVE: Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the white matter affecting immunocompromised patients that results from the cytolytic destruction of glial cells by the human neurotropic JC virus (JCV). According to one model, during the course of immunosuppression, JCV departs from its latent state in the kidney and after entering the brain, productively infects and destroys oligodendrocytes. The goal of this study was to test the hypothesis that JCV may reside in a latent state in a specific region of the brains of immunocompetent (non-PML) individuals without any neurological conditions. METHODS: Gene amplification was performed together with immunohistochemistry to examine the presence of JCV DNA sequences and expression of its genome in five distinct regions of the brain from seven immunocompetent non-PML individuals. RESULTS: Although no viral proteins were expressed in any of these cases, fragments of the viral DNA were present in various regions of normal brain. Laser-capture microdissection showed the presence of JCV DNA in oligodendrocytes and astrocytes, but not in neurons. INTERPRETATION: The detection of fragments of viral DNA in non-PML brain suggests that JCV has full access to all regions of the brain in immunocompetent individuals. Thus, should the immune system become impaired, the passing and/or the resident virus may gain the opportunity to express its genome and initiate its lytic cycle in oligodendrocytes. The brain as a site of JCV latency is a possibility.

Phosphofructokinases Axis Controls Glucose-Dependent mTORC1 Activation Driven by E2F1.

Cancer cells rely on mTORC1 activity to coordinate mitogenic signaling with nutrients availability for growth. Based on the metabolic function of E2F1, we hypothesize that glucose catabolism driven by E2F1 could participate on mTORC1 activation. Here, we demonstrate that glucose potentiates E2F1-induced mTORC1 activation by promoting mTORC1 translocation to lysosomes, a process that occurs independently of AMPK activation. We showed that E2F1 regulates glucose metabolism by increasing aerobic glycolysis and identified the PFKFB3 regulatory enzyme as an E2F1-regulated gene important for mTORC1 activation. Furthermore, PFKFB3 and PFK1 were found associated to lysosomes and we demonstrated that modulation of PFKFB3 activity, either by substrate accessibility or expression, regulates the translocation of mTORC1 to lysosomes by direct interaction with Rag B and subsequent mTORC1 activity. Our results support a model whereby a glycolytic metabolon containing phosphofructokinases transiently interacts with the lysosome acting as a sensor platform for glucose catabolism toward mTORC1 activity.

Oncogenic MYC Induces the Impaired Ribosome Biogenesis Checkpoint and Stabilizes p53 Independent of Increased Ribosome Content.

The role of MYC in regulating p53 stability as a function of increased ribosome biogenesis is controversial. On the one hand, it was suggested that MYC drives the overexpression of ribosomal proteins (RP)L5 and RPL11, which bind and inhibit HDM2, stabilizing p53. On the other, it has been proposed that increased ribosome biogenesis leads the consumption of RPL5/RPL11 into nascent ribosomes, reducing p53 levels and enhancing tumorigenesis. Here, we show that the components that make up the recently described impaired ribosome biogenesis checkpoint (IRBC) complex, RPL5, RPL11, and 5S rRNA, are reduced following MYC silencing. This leads to a rapid reduction in p53 protein half-life in an HDM2-dependent manner. In contrast, MYC induction leads to increased ribosome biogenesis and p53 protein stabilization. Unexpectedly, there is no change in free RPL5/RPL11 levels, but there is a striking increase in IRBC complex bound to HDM2. Our data support a cell-intrinsic tumor-suppressor response to MYC expression, which is presently being exploited to treat cancer. SIGNIFICANCE: Oncogenic MYC induces the impaired ribosome biogenesis checkpoint, which could be potentially targeted for cancer treatment.

The transcribed pseudogene RPSAP52 enhances the oncofetal HMGA2-IGF2BP2-RAS axis through LIN28B-dependent and independent let-7 inhibition.

One largely unknown question in cell biology is the discrimination between inconsequential and functional transcriptional events with relevant regulatory functions. Here, we find that the oncofetal HMGA2 gene is aberrantly reexpressed in many tumor types together with its antisense transcribed pseudogene RPSAP52. RPSAP52 is abundantly present in the cytoplasm, where it interacts with the RNA binding protein IGF2BP2/IMP2, facilitating its binding to mRNA targets, promoting their translation by mediating their recruitment on polysomes and enhancing proliferative and self-renewal pathways. Notably, downregulation of RPSAP52 impairs the balance between the oncogene LIN28B and the tumor suppressor let-7 family of miRNAs, inhibits cellular proliferation and migration in vitro and slows down tumor growth in vivo. In addition, high levels of RPSAP52 in patient samples associate with a worse prognosis in sarcomas. Overall, we reveal the roles of a transcribed pseudogene that may display properties of an oncofetal master regulator in human cancers.

1-Methoxy-canthin-6-one induces c-Jun NH2-terminal kinase-dependent apoptosis and synergizes with tumor necrosis factor-related apoptosis-inducing ligand activity in human neoplastic cells of hematopoietic or endodermal origin.

We investigated the effects of 1-methoxy-canthin-6-one, isolated from the medicinal plant Ailanthus altissima Swingle, on apoptosis in human leukemia (Jurkat), thyroid carcinoma (ARO and NPA), and hepatocellular carcinoma (HuH7) cell lines. Cultures incubated with the compound showed >50% of sub-G1 (hypodiploid) elements in flow cytometry analysis; the apoptosis-inducing activity was evident at <10 micromol/L and half-maximal at about 40 micromol/L 1-methoxy-canthin-6-one. The appearance of hypodiploid elements was preceded by mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and Smac/DIABLO and procaspase-3 cleavage. We subsequently investigated the effect of 1-methoxy-canthin-6-one in combination with human recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the four cell lines. Suboptimal concentrations (10 micromol/L 1-methoxy-canthin-6-one and 0.25 ng/mL TRAIL, respectively) of the two agents, unable to elicit apoptosis when used alone, induced mitochondrial depolarization, activation of caspase-3, and 45% to 85% of sub-G1 elements when added together to the cells. The synergism seemed to rely partly on the enhanced expression of TRAIL receptor 1 (TRAIL-R1; DR4), analyzed by immunofluorescence, by 1-methoxy-canthin-6-one. Cell incubation with 1-methoxy-canthin-6-one resulted in activating c-Jun NH2-terminal kinase (JNK), as revealed by Western blotting; induction of apoptosis and TRAIL-R1 up-regulation by 1-methoxy-canthin-6-one were >80% prevented by the addition of the JNK inhibitor (JNKI) SP600125JNKI, indicating that both effects were almost completely mediated by JNK activity. On the other hand, synergism with TRAIL was reduced by about 50%, suggesting that besides up-regulating TRAIL-R1, 1-methoxy-canthin-6-one could influence other factor(s) that participated in TRAIL-induced apoptosis. These findings indicate that 1-methoxy-canthin-6-one can represent a candidate for in vivo studies of monotherapies or combined antineoplastic therapies.

Apoptosis inhibition in cancer cells: a novel molecular pathway that involves BAG3 protein.

Stress-induced apoptosis regulates neoplasia pathogenesis and response to therapy. Indeed, cell transformation induces a stress response, that is overcome, in neoplastic cells, by alterations in apoptosis modulators; on the other hand, antineoplastic therapies largely trigger the apoptosis stress pathway, whose impairment results in resistance. Therefore, the study of the roles of apoptosis-modulating molecules in neoplasia development and response to therapy is of key relevance for our understanding of these processes. Among molecules that regulate apoptosis, a role is emerging for BAG3, a member of the BAG co-chaperone protein family. Proteins that share the BAG domain are characterized by their interaction with a variety of partners (heat shock proteins, steroid hormone receptors, Raf-1 and others), involved in regulating a number of cellular processes, including proliferation and apoptosis. BAG3, also known as CAIR-1 or Bis, forms a complex with the heat shock protein (Hsp) 70. This assists polypeptide folding, can mediate protein delivery to proteasome and is able to modulate apoptosis by interfering with cytochrome c release, apoptosome assembly and other events in the death process. It has been recently shown that, in human primary lymphoid and myeloblastic leukemias and other neoplastic cell types, BAG3 expression sustains cell survival and underlies resistance to therapy, through downmodulation of apoptosis. This review summarizes findings that assign an apoptotic role to BAG3 in some neoplastic cell types and identify the protein as a candidate target of therapy.

The antiapoptotic protein BAG3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand.

CONTEXT: We previously showed that BAG3 protein, a member of the BAG (Bcl-2-associated athanogene) co-chaperone family, modulates apoptosis in human leukemias. The expression of BAG3 in other tumor types has not been extensively investigated so far. OBJECTIVE: The objective of this study was to analyze BAG3 expression in thyroid neoplastic cells and investigate its influence in cell apoptotic response to TNF-related apoptosis-inducing ligand (TRAIL). DESIGN, SETTING, AND PATIENTS: We investigated BAG3 expression in human thyroid carcinoma cell lines, including NPA, and the effect of BAG3-specific small interfering RNA on TRAIL-induced apoptosis in NPA cells. Subsequently, we analyzed BAG3 expression in 30 benign lesions and 56 carcinomas from patients of the Naples Tumor Institute Fondazione Senatore Pascale. MAIN OUTCOME MEASURES: The main outcome measures were: analysis of BAG3 protein in NPA cells by Western blot and immunocytochemistry; analysis of apoptosis in TRAIL-stimulated NPA cells by flow cytometry; and evaluation of BAG3 expression in specimens from thyroid lesions by immunohistochemistry. RESULTS: BAG3 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downmodulation of its levels significantly (P < 0.0195) enhanced NPA cell apoptotic response to TRAIL. The protein was not detectable in 19 of 20 specimens of normal thyroid or goiters, whereas 54 of 56 analyzed carcinomas (15 follicular, 28 papillary, and 13 anaplastic) were clearly positive for BAG3 expression. CONCLUSIONS: BAG3 downmodulates the apoptotic response to TRAIL in human neoplastic thyroid cells. The protein is specifically expressed in thyroid carcinomas and not in normal thyroid tissue or goiter.

Inhibition of p63 transcriptional activity by p14ARF: functional and physical link between human ARF tumor suppressor and a member of the p53 family.

The ARF/MDM2/p53 pathway is a principal defense mechanism to protect the organism from uncontrolled effects of deregulated oncogenes. Oncogenes activate ARF, which interacts with and inhibits the ubiquitin ligase MDM2, resulting in p53 stabilization and activation. Once stabilized and activated, p53 can either induce or repress a wide array of different gene targets, which in turn can regulate cell cycle, DNA repair, and a number of apoptosis-related genes. Here we show that, unlike p53, p63, a member of the p53 family, directly interacts with p14(ARF). Through this interaction ARF inhibits p63-mediated transactivation and transrepression. In p63-transfected cells, ARF, which normally localizes into nucleoli, accumulates in the nucleoplasm. Based on these observations, we suggest that stimuli inducing p14(ARF) expression can, at the same time, activate p53 and impair p63 transcriptional activity, altering the pattern of p53 target gene expression. Here we show, for the first time, a physical and functional link between the p14(ARF) tumor suppressor protein and p63, a member of the p53 family.