ABSTRACT
The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.
Subject(s)
Antigen Presentation , Histocompatibility Antigens Class I/biosynthesis , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , T-Lymphocytes/metabolism , Animals , Cell Line, Tumor , Coculture Techniques , HEK293 Cells , Histocompatibility Antigens Class I/immunology , Host-Pathogen Interactions , Humans , Immunologic Surveillance , Influenza A virus/immunology , Influenza A virus/pathogenicity , Melanoma/immunology , Melanoma/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Ribosomal Proteins/genetics , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , Skin Neoplasms/immunology , Skin Neoplasms/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/virologyABSTRACT
Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.
Subject(s)
Antigens, CD20/genetics , Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Lymphoma, Non-Hodgkin/drug therapy , Rituximab/pharmacology , Animals , Antigens, CD20/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase 6 , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphoma, Non-Hodgkin/genetics , Lymphoma, Non-Hodgkin/immunology , Lymphoma, Non-Hodgkin/pathology , Mice, Inbred BALB C , Mice, SCID , RNA, Messenger/genetics , Tumor Cells, Cultured , Up-Regulation/drug effectsABSTRACT
Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of "specialized ribosomes", which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today.
Subject(s)
Eukaryota/genetics , Eukaryota/metabolism , Gene Expression Regulation , Protein Biosynthesis , Ribosomes/metabolism , Animals , Eukaryotic Cells , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Ribosomal Proteins/metabolismABSTRACT
Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Colorectal Neoplasms/drug therapy , DNA, Neoplasm/genetics , Drug Tolerance/genetics , Fluorouracil/pharmacology , Protein Biosynthesis/drug effects , Receptor, IGF Type 1/genetics , Cell Line, Tumor , Cell Survival/drug effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA Replication , DNA, Neoplasm/metabolism , Drug Resistance, Neoplasm/genetics , HCT116 Cells , Halogenation , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/metabolism , Ribosomes/drug effects , Ribosomes/genetics , Ribosomes/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Ribosomes are nanomachines essential for protein production in all living cells. Ribosome synthesis increases in cancer cells to cope with a rise in protein synthesis and sustain unrestricted growth. This increase in ribosome biogenesis is reflected by severe morphological alterations of the nucleolus, the cell compartment where the initial steps of ribosome biogenesis take place. Ribosome biogenesis has recently emerged as an effective target in cancer therapy, and several compounds that inhibit ribosome production or function, killing preferentially cancer cells, have entered clinical trials. Recent research indicates that cells express heterogeneous populations of ribosomes and that the composition of ribosomes may play a key role in tumorigenesis, exposing novel therapeutic opportunities. Here, we review recent data demonstrating that ribosome biogenesis is a promising druggable pathway in cancer therapy, and discuss future research perspectives.
Subject(s)
Antineoplastic Agents/pharmacology , Ribosomes/drug effects , Ribosomes/metabolism , Humans , Molecular Targeted Therapy/methods , Neoplasms/drug therapy , Neoplasms/pathology , RNA, Ribosomal/metabolismABSTRACT
In mammals, FOXO transcriptional factors form a family of four members (FOXO1, 3, 4, and 6) involved in the modulation proliferation, apoptosis, and carcinogenesis. The role of the FOXO family in breast cancer remains poorly elucidated. According to the cellular context and the stage of the disease, FOXOs can have opposite effects on carcinogenesis. To study the role of FOXOs in breast carcinogenesis in more detail, we examined their expression in normal tissues, breast cell lines, and a large series of breast tumours of human origin. We found a very low physiological level of FOXO6 expression in normal adult tissues and high levels of expression in foetal brain. FOXO gene expressions fluctuate specifically in breast cancer cells compared to normal cells, suggesting that these genes may have different roles in breast carcinogenesis. For the first time, we have shown that, among the various FOXO genes, only FOXO6 was frequently highly overexpressed in breast cell lines and tumours. We also found that inhibition of the endogenous expression of FOXO6 by a specific siRNA inhibited the growth of the human breast cell lines MDA-MB-468 and HCC-38. FACS and Western blot analysis showed that inhibition of endogenous expression of FOXO6 induced accumulation of cells in G0/G1 phase of the cell cycle, but not apoptosis. These results tend to demonstrate that the overexpression of the human FOXO6 gene that we highlighted in the breast tumors stimulates breast carcinogenesis by activating breast cancer cell proliferation.
ABSTRACT
5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug in colorectal cancer. Previous studies showed that 5-FU modulates RNA metabolism and mRNA expression. In addition, it has been reported that 5-FU incorporates into the RNAs constituting the translational machinery and that 5-FU affects the amount of some mRNAs associated with ribosomes. However, the impact of 5-FU on translational regulation remains unclear. Using translatome profiling, we report that a clinically relevant dose of 5-FU induces a translational reprogramming in colorectal cancer cell lines. Comparison of mRNA distribution between polysomal and non-polysomal fractions in response to 5-FU treatment using microarray quantification identified 313 genes whose translation was selectively regulated. These regulations were mostly stimulatory (91%). Among these genes, we showed that 5-FU increases the mRNA translation of HIVEP2, which encodes a transcription factor whose translation in normal condition is known to be inhibited by mir-155. In response to 5-FU, the expression of mir-155 decreases thus stimulating the translation of HIVEP2 mRNA. Interestingly, the 5-FU-induced increase in specific mRNA translation was associated with reduction of global protein synthesis. Altogether, these findings indicate that 5-FU promotes a translational reprogramming leading to the increased translation of a subset of mRNAs that involves at least for some of them, miRNA-dependent mechanisms. This study supports a still poorly evaluated role of translational control in drug response.
Subject(s)
Colonic Neoplasms/therapy , Colorectal Neoplasms/therapy , Fluorouracil/therapeutic use , MicroRNAs/genetics , RNA, Messenger/genetics , Cellular Reprogramming , Colonic Neoplasms/genetics , Colorectal Neoplasms/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , HCT116 Cells , HT29 Cells , Humans , Protein Biosynthesis/drug effects , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
PURPOSE: Whereas post-radiation therapy overreactions (OR) represent a clinical and societal issue, there is still no consensual radiobiological endpoint to predict clinical radiosensitivity. Since 2003, skin biopsy specimens have been collected from patients treated by radiation therapy against different tumor localizations and showing a wide range of OR. Here, we aimed to establish quantitative links between radiobiological factors and OR severity grades that would be relevant to radioresistant and genetic hyperradiosensitive cases. METHODS AND MATERIALS: Immunofluorescence experiments were performed on a collection of skin fibroblasts from 12 radioresistant, 5 hyperradiosensitive, and 100 OR patients irradiated at 2 Gy. The numbers of micronuclei, γH2AX, and pATM foci that reflect different steps of DNA double-strand breaks (DSB) recognition and repair were assessed from 10 minutes to 24 hours after irradiation and plotted against the severity grades established by the Common Terminology Criteria for Adverse Events and the Radiation Therapy Oncology Group. RESULTS: OR patients did not necessarily show a gross DSB repair defect but a systematic delay in the nucleoshuttling of the ATM protein required for complete DSB recognition. Among the radiobiological factors, the maximal number of pATM foci provided the best discrimination among OR patients and a significant correlation with each OR severity grade, independently of tumor localization and of the early or late nature of reactions. CONCLUSIONS: Our results are consistent with a general classification of human radiosensitivity based on 3 groups: radioresistance (group I); moderate radiosensitivity caused by delay of nucleoshuttling of ATM, which includes OR patients (group II); and hyperradiosensitivity caused by a gross DSB repair defect, which includes fatal cases (group III).
Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Nucleus/metabolism , DNA Breaks, Double-Stranded , Histones/metabolism , Radiation Injuries/classification , Radiation Tolerance/physiology , Skin/radiation effects , Analysis of Variance , Ataxia Telangiectasia Mutated Proteins/genetics , Biopsy , Cell Line , DNA Repair , Fibroblasts/radiation effects , Humans , Micronucleus Tests/methods , Phosphorylation , Radiation Injuries/metabolism , Radiation Injuries/pathology , Radiation Tolerance/genetics , Skin/pathology , Time FactorsABSTRACT
4.1R pre-mRNA alternative splicing results in multiple mRNA and protein isoforms that are expressed in virtually all tissues. More specifically, isoforms containing the alternative exon 17a, are exclusively expressed in muscle tissues. In this report, we show that these isoforms are preferentially present in the myoplasm of fast myofibres. 4.1R epitopes are also found at the sarcolemma of both slow and fast myofibres in normal muscle. Interestingly, they are absent from dystrophin-deficient sarcolemma of DMD muscle, and colocalize with partially expressed dystrophin in BMD muscle. We also show that alternative splicing of exons 16 and 17a is regulated during muscle differentiation in an asynchronous fashion, with an early inclusion of exon 16 in forming myotubes, and a late inclusion of exon 17a. Consistently, Western blot analysis led to characterize mainly an approximately 96/98-kDa doublet bearing exons 16-17a-encoding peptide, exclusively occurring in the differentiated muscle.
Subject(s)
Alternative Splicing , Blood Proteins/genetics , Microtubule-Associated Proteins/genetics , Muscle, Skeletal/metabolism , Muscular Dystrophies/genetics , Amino Acid Sequence , Base Sequence , Blood Proteins/metabolism , Cell Line , Cytoskeletal Proteins , DNA, Complementary/genetics , Humans , Membrane Proteins , Microtubule-Associated Proteins/metabolism , Molecular Sequence Data , Muscular Dystrophies/metabolism , RNA Precursors/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
As glucose is a mandatory nutrient for cell proliferation and renewal, it is suspected that glucose microenvironment is sensed by all cell types to regulate angiogenesis. Several glucose-sensing components have been partially described to respond to high glucose levels. However, little is known about the response to low glucose. Here, we used well-differentiated isolated normal rat renal tubules under normal oxygenation conditions to assess the angiogenic response to low glucose. In apparent paradox, but confirming observations made separately in other models, high glucose but also low glucose increased mRNA level of vascular endothelial growth factor A (VEGFA). A subset of mRNAs including hypoxia-inducible factor 1A (HIF1A), angiopoietin receptor (TIE-2), and VEGF receptor 2 (FLK1) were similarly glucose-sensitive and responded to low glucose by increased stability independently of HIF1A and HIF2A proteins. These results contribute to gain some insights as to how normal cells response to low glucose may play a role in the tumor microenvironment.
ABSTRACT
BRCA1 acts as a tumor suppressor gene, and germ-line mutations in this gene are found in a large proportion of families with breast and ovarian cancers. The BRCA1 protein has been implicated in several cellular processes, such as transcription regulation, DNA responses to DNA damage signals, cell cycle control, and apoptosis. Apoptosis plays a critical role in radiation- and chemotherapy-induced cytotoxicity, and its impairment contributes to resistance to tumor treatments. In an attempt to elucidate the role of BRCA1 in apoptosis, we examined the response to chemotherapeutic drugs of cells expressing physiological levels of BRCA1 protein. We showed that chemotherapy-induced apoptosis leads to a caspase-mediated cleavage of BRCA1. We then showed that the BRCA1-p90 cleavage product is mainly localized in the cytoplasm. Finally, we demonstrated that cancer-associated mutations affecting the BRCT tandem repeat abolish its pro-apoptotic function. The data presented here provide new insight into the role of endogenous BRCA1 as a mediator of apoptosis and show that BRCA1 functions as a molecular determinant of response to a range of cytotoxic chemotherapeutic agents.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , BRCA1 Protein/metabolism , Caspases/metabolism , Breast Neoplasms , Cell Line, Tumor , Cytoplasm/metabolism , Female , HeLa Cells , Humans , Jurkat CellsSubject(s)
AMP-Activated Protein Kinases/metabolism , Acetyl-CoA Carboxylase/metabolism , Spindle Poles/metabolism , AMP-Activated Protein Kinases/chemistry , Acetyl-CoA Carboxylase/analysis , Benzimidazoles/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Centrosome/metabolism , Cytokinesis/drug effects , Humans , Mitosis , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Thiophenes/pharmacology , Polo-Like Kinase 1ABSTRACT
BRCA1 has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair, cell cycle control, and apoptosis. We identified poly(A)-binding protein 1 (PABP) as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by in vitro assays and coimmunoprecipitation in mammalian cells. Endogenous interaction between BRCA1 and PABP was also observed. This interaction was abolished by BRCA1 cancer-associated mutations, suggesting that it may be physiologically relevant. Deletion mapping demonstrated that the RNA recognition motifs 1-4 region of PABP is required to mediate the interaction with BRCA1. To understand the biological function of the BRCA1-PABP complex, we sought to determine whether BRCA1 is a modulator of translation. We showed here that inhibition of endogenous BRCA1 using a small interfering RNA-based approach decreased protein synthesis. Conversely, overexpression of BRCA1 activated translation. Using a RNA transfection approach, we clearly showed that BRCA1 modulates translation, independently of any transcriptional activity. The data presented here suggest that BRCA1 modulates protein synthesis via its interaction with PABP, providing a novel mechanism by which BRCA1 may exert its tumor suppressor function.