eIF2Bδ blocks the integrated stress response and maintains eIF2B activity and cancer metastasis by overexpression in breast cancer stem cells.

Breast cancer (BC) metastasis involves cancer stem cells (CSCs) and their regulation by micro-RNAs (miRs), but miR targeting of the translation machinery in CSCs is poorly explored. We therefore screened miR expression levels in a range of BC cell lines, comparing non-CSCs to CSCs, and focused on miRs that target translation and protein synthesis factors. We describe a unique translation regulatory axis enacted by reduced expression of miR-183 in breast CSCs, which we show targets the eIF2Bδ subunit of guanine nucleotide exchange factor eIF2B, a regulator of protein synthesis and the integrated stress response (ISR) pathway. We report that reduced expression of miR-183 greatly increases eIF2Bδ protein levels, preventing strong induction of the ISR and eIF2α phosphorylation, by preferential interaction with P-eIF2α. eIF2Bδ overexpression is essential for BC cell invasion, metastasis, maintenance of metastases, and breast CSC expansion in animal models. Increased expression of eIF2Bδ, a site of action of the drug ISRIB that also prevents ISR signaling, is essential for breast CSC maintenance and metastatic capacity.

Translational regulation of TFH cell differentiation and autoimmune pathogenesis.

Little is known regarding T cell translational regulation. We demonstrate that T follicular helper (TFH) cells use a previously unknown mechanism of selective messenger RNA (mRNA) translation for their differentiation, role in B cell maturation, and in autoimmune pathogenesis. We show that TFH cells have much higher levels of translation factor eIF4E than non-TFH CD4+ T cells, which is essential for translation of TFH cell fate-specification mRNAs. Genome-wide translation studies indicate that modest down-regulation of eIF4E activity by a small-molecule inhibitor or short hairpin RN impairs TFH cell development and function. In mice, down-regulation of eIF4E activity specifically reduces TFH cells among T helper subtypes, germinal centers, B cell recruitment, and antibody production. In experimental autoimmune encephalomyelitis, eIF4E activity down-regulation blocks TFH cell participation in disease pathogenesis while promoting rapid remission and spinal cord remyelination. TFH cell development and its role in autoimmune pathogenesis involve selective mRNA translation that is highly druggable.

Targeting eIF4F translation initiation complex with SBI-756 sensitises B lymphoma cells to venetoclax.

BACKGROUND: The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL). METHODS: We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex. RESULTS: Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis. CONCLUSIONS: Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.

Neurons Release Serine to Support mRNA Translation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.

Translation Regulation by eIF2α Phosphorylation and mTORC1 Signaling Pathways in Non-Communicable Diseases (NCDs).

Non-communicable diseases (NCDs) are medical conditions that, by definition, are non-infectious and non-transmissible among people. Much of current NCDs are generally due to genetic, behavioral, and metabolic risk factors that often include excessive alcohol consumption, smoking, obesity, and untreated elevated blood pressure, and share many common signal transduction pathways. Alterations in cell and physiological signaling and transcriptional control pathways have been well studied in several human NCDs, but these same pathways also regulate expression and function of the protein synthetic machinery and mRNA translation which have been less well investigated. Alterations in expression of specific translation factors, and disruption of canonical mRNA translational regulation, both contribute to the pathology of many NCDs. The two most common pathological alterations that contribute to NCDs discussed in this review will be the regulation of eukaryotic initiation factor 2 (eIF2) by the integrated stress response (ISR) and the mammalian target of rapamycin complex 1 (mTORC1) pathways. Both pathways integrally connect mRNA translation activity to external and internal physiological stimuli. Here, we review the role of ISR control of eIF2 activity and mTORC1 control of cap-mediated mRNA translation in some common NCDs, including Alzheimer's disease, Parkinson's disease, stroke, diabetes mellitus, liver cirrhosis, chronic obstructive pulmonary disease (COPD), and cardiac diseases. Our goal is to provide insights that further the understanding as to the important role of translational regulation in the pathogenesis of these diseases.

Binding of a viral IRES to the 40S subunit occurs in two successive steps mediated by eS25.

The mechanism for how internal ribosome entry sites (IRESs) recruit ribosomes to initiate translation of an mRNA is not completely understood. We investigated how a 40S subunit was recruited by the cricket paralysis virus intergenic region (CrPV IGR) IRES to form a stable 40S-IRES complex. Kinetic binding studies revealed that formation of the complex between the CrPV IGR and the 40S subunit consisted of two-steps: an initial fast binding step of the IRES to the 40S ribosomal subunit, followed by a slow unimolecular reaction consistent with a conformational change that stabilized the complex. We further showed that the ribosomal protein S25 (eS25), which is required by functionally and structurally diverse IRESs, impacts both steps of the complex formation. Mutations in eS25 that reduced CrPV IGR IRES activity either decreased 40S-IRES complex formation, or increased the rate of the conformational change that was required to form a stable 40S-IRES complex. Our data are consistent with a model in which eS25 facilitates initial binding of the CrPV IGR IRES to the 40S while ensuring that the conformational change stabilizing the 40S-IRES complex does not occur prematurely.

Inflammatory Breast Cancer Promotes Development of M2 Tumor-Associated Macrophages and Cancer Mesenchymal Cells through a Complex Chemokine Network.

Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer that displays profound cancer stem cell (CSC) and mesenchymal features that promote rapid metastasis. Another hallmark of IBC is high infiltration of M2 tumor-associated (immune-suppressing) macrophages. The molecular mechanism that drives these IBC phenotypes is not well understood. Using patient breast tumor specimens, breast cancer cell lines, and a patient-derived xenograft model of IBC, we demonstrate that IBC strongly expresses IL8 and growth-regulated oncogene (GRO) chemokines that activate STAT3, which promotes development of high levels of CSC-like cells and a mesenchymal phenotype. We also show that IBC expresses high levels of many monocyte recruitment and macrophage polarization factors that attract and differentiate monocytes into tumor-promoting, immune-suppressing M2-like macrophages. The M2 macrophages in turn were found to secrete high levels of IL8 and GRO chemokines, thereby creating a feed-forward chemokine loop that further drives an IBC epithelial-to-mesenchymal transition. Our study uncovers an intricate IBC-initiated autocrine-paracrine signaling network between IBC cells and monocytes that facilitates development of this highly aggressive form of breast cancer. SIGNIFICANCE: This study uncovers a signaling network in which IBC cells commandeer macrophages to become tumor-promoting, and they in turn drive IBC cells to be more cancer stem-like, mesenchymal, and aggressive.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3360/F1.large.jpg.

Quality Improvement Initiative Reduces the Occurrence of Complications in Peripherally Inserted Central Catheters.

Peripherally inserted central catheters (PICCs) are commonly used to access the central venous system. However, central vascular access devices are associated with a risk of complications, which may include infection, thrombosis, occlusion, or malposition. The vascular access team of 1 midwestern hospital used a quality improvement initiative to reduce the occurrence of complications associated with PICCs. A secondary aim was to reduce the number of alteplase (Cathflo Activase; Genentech, South San Francisco, CA) doses administered. After reviewing current evidence, the vascular access team employed the Plan-Do-Study-Act cycle to document and implement changes in practice. By using a team initiative to investigate this issue, complication rates decreased and patient satisfaction improved. The thrombosis rate decreased by 67%, occlusions by 75%, and alteplase use by 87%. No infections occurred during this study.

Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The HTLV-1 basic leucine zipper protein (HBZ) is expressed in all cases of ATL and is directly associated with virus pathogenicity. The two isoforms of the HBZ protein are synthesized from antisense messenger RNAs (mRNAs) that are either spliced (sHBZ) or unspliced (usHBZ) versions of the HBZ transcript. The sHBZ and usHBZ mRNAs have entirely different 5'untranslated regions (5'UTR) and are differentially expressed in cells, with the sHBZ protein being more abundant. Here, we show that differential expression of the HBZ isoforms is regulated at the translational level. Translation initiation of the usHBZ mRNA relies on a cap-dependent mechanism, while the sHBZ mRNA uses internal initiation. Based on the structural data for the sHBZ 5'UTR generated by SHAPE in combination with 5' and 3' deletion mutants, the minimal region harboring IRES activity was mapped to the 5'end of the sHBZ mRNA. In addition, the sHBZ IRES recruited the 40S ribosomal subunit upstream of the initiation codon, and IRES activity was found to be dependent on the ribosomal protein eS25 and eIF5A.

Foxp1 Negatively Regulates T Follicular Helper Cell Differentiation and Germinal Center Responses by Controlling Cell Migration and CTLA-4.

T follicular helper (Tfh) cells play an essential role in the formation of germinal centers (GC) and generation of high-affinity Abs. The homing of activated CD4+ T cells into B cell follicles and the involvement of key costimulatory and coinhibitory molecules are critical in controlling both the initiation and the magnitude of GC responses. Meanwhile, studies have shown that a high number of single clone B cells leads to intraclonal competition, which inhibits the generation of high-affinity Abs. Our previous work has shown that transcription factor Foxp1 is a critical negative regulator of Tfh cell differentiation. In this study, we report that the deletion of Foxp1 leads to a high proportion of activated CD4+ T cells homing into B cell follicles with faster kinetics, resulting in earlier GC formation. In addition, we show that Foxp1-deficient Tfh cells restore the generation of high-affinity Abs when cotransferred with high numbers of single clone B cells. We find that Foxp1 regulates the expression levels of cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) in activated CD4+ T cells and that Ctla4 is a direct Foxp1 target. Finally, we demonstrate that CTLA-4 expression on conventional CD4+ T cells plays a cell-intrinsic role in Tfh cell differentiation in vivo, and CTLA-4 blockade helps abolish the intraclonal competition of B cells in generating high-affinity Abs.

Translation initiation factors and their relevance in cancer.

Deregulation of several translation initiation factors occurs in numerous types of cancers. Translation initiation factors are not merely ancillary players in cancer development and progression, but rather, they are key participants in cellular transformation and tumor development. In fact, the altered expression of translation initiation factors is involved in cancer cell survival, metastasis and tumor angiogenesis. Although the exact mechanisms remain to be fully characterized, translation initiation factors comprise novel targets for pharmacologic intervention. Here we review the most recently established roles of initiation factors in cancer development and progression, as well as unique methods used to study translational regulation.

Cap-Independent Translational Control of Carcinogenesis.

Translational regulation has been shown to play an important role in cancer and tumor progression. Despite this fact, the role of translational control in cancer is an understudied and under appreciated field, most likely due to the technological hurdles and paucity of methods available to establish that changes in protein levels are due to translational regulation. Tumors are subjected to many adverse stress conditions such as hypoxia or starvation. Under stress conditions, translation is globally downregulated through several different pathways in order to conserve energy and nutrients. Many of the proteins that are synthesized during stress in order to cope with the stress use a non-canonical or cap-independent mechanism of initiation. Tumor cells have utilized these alternative mechanisms of translation initiation to promote survival during tumor progression. This review will specifically discuss the role of cap-independent translation initiation, which relies on an internal ribosome entry site (IRES) to recruit the ribosomal subunits internally to the messenger RNA. We will provide an overview of the role of IRES-mediated translation in cancer by discussing the types of genes that use IRESs and the conditions under which these mechanisms of initiation are used. We will specifically focus on three well-studied examples: Apaf-1, p53, and c-Jun, where IRES-mediated translation has been demonstrated to play an important role in tumorigenesis or tumor progression.

Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis.

Detection of human parechovirus (HPeV)-3 in spinal fluid specimens from pediatric patients in the Chicago area.

BACKGROUND: The human parechoviruses (HPeV) have recently been recognized as important viral pathogens causing various illnesses including sepsis and meningitis in children. However, data from the United States is limited. OBJECTIVES: To better understand the epidemiology of HPeV in the United States and its role in pediatric disease through detection and typing of the virus in cerebrospinal fluid specimens. STUDY DESIGN: Four hundred and twenty-one spinal fluid samples collected from 373 patients ranging in age from 1 day to 18 years were tested using a real-time reverse transcription-PCR assay. The specimens were originally collected for routine viral and bacterial testing to assist in the diagnosis of meningitis or sepsis. Amplification products of the VP1 region in the virus genome were sequenced to identify the parechovirus type. RESULTS: Ten positive specimens were identified from 10 different patients. All ten samples were typed as HPeV3 and were negative for bacteria by culture, and for enterovirus and herpes simplex virus by PCR. All of the HPeV3-infected patients were young infants ranging in age from 6 to 59 days. Infants in whom HPeV3 was detected had significantly decreased peripheral white blood cell counts. Positive specimens were all from the summer and early fall. CONCLUSIONS: HPeV3 infection of the central nervous system is found in very young infants in certain years during the summer and early fall, and is associated with leukopenia. Real-time RT-PCR is an effective tool for rapid detection of these infections, and could help prevent unnecessary hospitalization and antibiotic use in HPeV infected infants. More widespread use of this tool in diagnosing HPeV infection would aid in further clarifying the prevalence of this disease in the United States.