Abstract：Genomic instability has been observed in essentially all sporadic carcinomas. Here we use Drosophila epithelial cells to address the role of chromosomal instability in cancer development as they have proved useful for elucidating the molecular mechanisms underlying tumorigenic growth. We first show that chromosomal instability leads to an apoptotic response. Interestingly, this response is p53 independent, as opposed to mammalian cells, and depends on the activation of the c-Jun N-terminal kinase (JNK) signaling cascade. When prevented from undergoing programmed cell death (PCD), chromosomal instability induces neoplasic overgrowth. These tumor-like tissues are able to grow extensively and metastasize when transplanted into the abdomen of adult hosts. Detailed analysis of the tumors allows us to identify a delaminating cell population as the critical one in driving tumorigenesis. Cells loose their apical–basal polarity, mislocalize DE-cadherin, and delaminate from the main epithelium. A JNK-dependent transcriptional program is activated specifically in delaminating cells and drives nonautonomous tissue overgrowth, basement membrane degradation, and invasiveness. These findings unravel a general and rapid tumorigenic potential of genomic instability, as opposed to its proposed role as a source of mutability to select specific tumor-prone aneuploid cells, and open unique avenues toward the understanding of the role of genomic instability in human cancer.
Local Ablative Therapy of Oligoprogressive Disease Prolongs Disease Control by Tyrosine Kinase Inhibitors in Oncogene-Addicted Non–Small-Cell Lung Cancer
Introduction: Many patients with oncogene-driven non–small-cell lung cancer (NSCLC) treated with tyrosine kinase inhibitors experience limited sites of disease progression. This study investigated retrospectively the benefits of local ablative therapy (LAT) to central nervous system (CNS) and/or limited systemic disease progression and continuation of crizotinib or erlotinib in patients with metastatic ALK gene rearrangement (ALK+) or EGFR-mutant (EGFR-MT) NSCLC, respectively.
Methods: Patients with metastatic ALK+ NSCLC treated with crizotinib (n = 38) and EGFR-MT NSCLC treated with erlotinib (n = 27) were identified at a single institution. Initial response to the respective kinase inhibitors, median progression-free survival (PFS1), and site of first progression were recorded. A subset of patients with either nonleptomeningeal CNS and/or four sites or fewer of extra-CNS progression (oligoprogressive disease) suitable for LAT received either radiation or surgery to these sites and continued on the same tyrosine kinase inhibitors. The subsequent median progression-free survival from the time of first progression (PFS2) and pattern of progression were recorded.
Results: Median progression-free survival in ALK+ patients on crizotinib was 9.0 months, and 13.8 months for EGFR-MT patients on erlotinib. Twenty-five of 51 patients (49%) who progressed were deemed suitable for local therapy (15 ALK+, 10 EGFR-MT; 24 with radiotherapy, one with surgery) and continuation of the same targeted therapy. Post-LAT, 19 of 25 patients progressed again, with median PFS2 of 6.2 months.
Discussion: Oncogene-addicted NSCLC with CNS and/or limited systemic disease progression (oligoprogressive disease) on relevant targeted therapies is often suitable for LAT and continuation of the targeted agent, and is associated with more than 6 months of additional disease control.
Adult Pancreas Side Population Cells Expand after β Cell Injury and Are a Source of Insulin-Secreting Cells
Pancreas stem cells are a potential source of insulin-producing β cells for the therapy of diabetes. In adult tissues the 'side population' (SP) of cells that effluxes the DNA binding dye Hoechst 33342 through ATP-binding cassette transporters has stem cell properties. We hypothesised therefore that the SP would expand in response to β cell injury and give rise to functional β cells. SP cells were flow sorted from dissociated pancreas cells of adult mice, analysed for phenotype and cultured with growth promoting and differentiation factors before analysis for hormone expression and glucose-stimulated insulin secretion. SP cell number and colony forming potential (CFP) increased significantly in models of type diabetes, and after partial pancreatectomy, in the absence of hyperglycaemia. SP cells, ～1% of total pancreas cells at 1 week of age, were enriched >10-fold for CFP compared to non-SP cells. Freshly isolated SP cells contained no insulin protein or RNA but expressed the homeobox transcription factor Pdx1 required for pancreas development and β cell function. Pdx1, along with surface expression of CD326 (Ep-Cam), was a marker of the colony forming and proliferation potential of SP cells. In serum-free medium with defined factors, SP cells proliferated and differentiated into islet hormone-expressing cells that secreted insulin in response to glucose. Insulin expression was maintained when tissue was transplanted within vascularised chambers into diabetic mice. SP cells in the adult pancreas expand in response to β cell injury and are a source of β cell progenitors with potential for the treatment of diabetes.
Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful1, 2, 3, a minority of individuals naturally develop these antibodies after many years of infection4, 5, 6, 7. How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1–infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly under-represented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.
Valosin-containing protein (VCP, also called p97) is an essential and highly conserved adenosine triphosphate-dependent chaperone implicated in a wide range of cellular processes in eukaryotes, and mild VCP mutations can cause severe neurodegenerative disease. Here we show that mammalian VCP is trimethylated on Lys315 in a variety of cell lines and tissues, and that the previously uncharacterized protein METTL21D (denoted here as VCP lysine methyltransferase, VCP-KMT) is the responsible enzyme. VCP methylation was abolished in three human VCP-KMT knockout cell lines generated with zinc-finger nucleases. Interestingly, VCP-KMT was recently reported to promote tumour metastasis, and indeed, VCP-KMT-deficient cells displayed reduced growth rate, migration and invasive potential. Finally, we present data indicating that VCP-KMT, calmodulin-lysine methyltransferase and eight uncharacterized proteins together constitute a novel human protein methyltransferase family. The present work provides new insights on protein methylation and its links to human disease.
Translational repression and mRNA degradation are two major mechanisms for post-transcriptional regulation of gene expression. The detailed relationship between these two processes is not yet well established. Zinc-finger antiviral protein (ZAP) inhibits the replication of certain viruses, including human immunodeficiency virus 1, by binding directly to specific viral mRNAs and recruiting cellular mRNA degradation machinery to degrade the target mRNA. Here, we report that ZAP also inhibits the translation of target mRNAs by interfering with the interaction between translational initiation factors eIF4G and eIF4A. Furthermore, we provide evidence that translational repression is required for mRNA degradation and that blocking the degradation of target mRNAs does not affect ZAP-mediated translational repression. We conclude that ZAP can both repress translation and promote degradation of target mRNA, and that translational repression precedes and is required for mRNA degradation.
PIWI-interacting RNAs (piRNAs) silence transposons to maintain genome integrity in animal germ lines1, 2, 3, 4. piRNAs are classified as primary and secondary piRNAs, depending on their biogenesis machinery5, 6, 7, 8, 9, 10. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters in the genome through the primary processing pathway5, 8, 9, 10. Although the existence of a ribonuclease participating in this pathway has been predicted, its molecular identity remained unknown. Here we show that Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily member11, is an endoribonuclease essential for primary piRNA biogenesis. We solved the crystal structure of Drosophila melanogaster Zuc (DmZuc) at 1.75?? resolution. The structure revealed that DmZuc has a positively charged, narrow catalytic groove at the dimer interface, which could accommodate a single-stranded, but not a double-stranded, RNA. DmZuc and the mouse homologue MmZuc (also known as Pld6 and MitoPLD)12, 13, 14 showed endoribonuclease activity for single-stranded RNAs in vitro. The RNA cleavage products bear a 5′-monophosphate group, a hallmark of mature piRNAs. Mutational analyses revealed that the conserved active-site residues of DmZuc are critical for the ribonuclease activity in vitro, and for piRNA maturation and transposon silencing in vivo. We propose a model for piRNA biogenesis in animal germ lines, in which the Zuc endoribonuclease has a key role in primary piRNA maturation.
Study Objective: To determine the hormonal effects of reducing sleep duration under controlled feeding conditions. Design: Randomized, crossover study. Setting: Inpatient. Participants: Twenty-seven normal weight, 30- to 45-yr-old men and women habitually sleeping 7-9 hr/night. Intervention: Participants were studied under two sleep conditions: short (4 hr in bed) or habitual (9 hr in bed) sleep. A controlled diet was provided for each 4-day study period. Measurements and Results: Fasting blood samples were obtained daily and frequent blood samples were obtained throughout day 4. The main outcomes measures included glucose, insulin, leptin, ghrelin, adiponectin, total glucagon-like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) concentrations. Body weights were reduced by 2.2 ± 0.4 lb and 1.7 ± 0.4 lb during the habitual and short sleep phases, respectively (both P < 0.0001). There was no effect of sleep duration on glucose, insulin, and leptin profiles (all P > 0.05). Ghrelin and GLP-1 responses differed by sex. Short sleep increased fasting (P = 0.054) and morning (08:00-12:00) (P = 0.042) total ghrelin in men but not women. The reverse was observed for GLP-1: afternoon levels (12:30-19:00) were lower (P = 0.016) after short sleep compared with habitual sleep in women but not men. Conclusions: These data suggest that, in the context of negative energy balance, short sleep does not lead to a state of increased insulin resistance, but may predispose to overeating via separate mechanisms in men and women.
Exosomes Mediate the Cytoprotective Action of Mesenchymal Stromal Cells on Hypoxia-Induced Pulmonary Hypertension
Background—Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of pro-inflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension (HPH). Mesenchymal stromal cell (MSC) transplantation inhibits lung inflammation, vascular remodeling and right heart failure, and reverses HPH in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which MSCs are protective in HPH. Methods and Results—We fractionated mouse MSC-conditioned media to identify the biologically-active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of pro-inflammatory and pro-proliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of HPH. Intravenous delivery of MSC-derived exosomes (MEX) inhibited vascular remodeling and HPH, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA whose expression is decreased in human PH. MEX produced by human umbilical cord MSCs inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells demonstrating a direct effect of MEX on hypoxic vascular cells. Conclusions—This study indicates that MEX exert a pleiotropic protective effect on the lung and inhibit PH through suppression of hyperproliferative pathways, including STAT-3 mediated signaling induced by hypoxia.
Acute Epstein-Barr virus (EBV) infection results in an unusually robust CD8+ T cell response in young adults. Based on mouse studies, such a response would be predicted to result in attrition of preexisting memory to heterologous infections like influenza A (Flu) and cytomegalovirus (CMV). Furthermore, many studies have attempted to define the lymphocytosis that occurs during acute EBV infection in humans, but it is unclear whether bystander T cells contribute to it. To address these issues, we performed a longitudinal prospective study of primary EBV infection in humans. During acute EBV infection, both preexisting CMV- and Flu-specific memory CD8+ T cells showed signs of bystander activation, including up-regulation of granzyme B. However, they generally did not expand, suggesting that the profound CD8+ lymphocytosis associated with acute EBV infection is composed largely of EBV-specific T cells. Importantly, the numbers of CMV- and Flu-specific T cells were comparable before and after acute EBV infection. The data support the concept that, in humans, a robust CD8+ T cell response creates a new memory CD8+ T cell niche without substantially depleting preexisting memory for heterologous infections.