ABSTRACT
Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.
Subject(s)
C9orf72 Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Dipeptides/metabolism , Proteolysis , Small Molecule Libraries/pharmacology , Animals , Cell Line , Codon, Initiator/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA Repeat Expansion/genetics , Disease Models, Animal , Drosophila/drug effects , Frontotemporal Dementia/pathology , HEK293 Cells , High-Throughput Screening Assays , Humans , Induced Pluripotent Stem Cells/pathology , Isoquinolines/pharmacology , Longevity/drug effects , Motor Neurons/drug effects , Motor Neurons/pathology , Protein Biosynthesis/drug effects , Proteolysis/drug effects , RNA Interference , Sulfonamides/pharmacologyABSTRACT
Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.
Subject(s)
Breast Neoplasms/diet therapy , Breast Neoplasms/drug therapy , Diet Therapy/methods , Fasting/physiology , Fulvestrant/therapeutic use , Animals , Biological Factors/blood , Breast Neoplasms/pathology , Diet, Healthy/methods , Disease Models, Animal , Disease Progression , Drug Resistance, Neoplasm/drug effects , Early Growth Response Protein 1/metabolism , Female , Fulvestrant/administration & dosage , Humans , Insulin/blood , Insulin-Like Growth Factor I/metabolism , Leptin/blood , MCF-7 Cells , Mice, Inbred NOD , Mice, SCID , PTEN Phosphohydrolase/metabolism , Piperazines/administration & dosage , Piperazines/therapeutic use , Pyridines/administration & dosage , Pyridines/therapeutic use , Receptors, Estrogen , Receptors, Progesterone , Tamoxifen/adverse effects , Tamoxifen/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m6A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m6A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.
Subject(s)
RNA-Binding Proteins , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Pliability , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Molecular ConformationABSTRACT
LncRNAs play crucial roles in cellular processes and their regulatory effects in the adult brain and neural stem cells (NSCs) remain to be entirely characterized. We report that 10 lncRNAs (LincENC1, FABL, lincp21, HAUNT, PERIL, lincBRN1a, lincBRN1b, HOTTIP, TUG1 and FENDRR) are expressed during murine NSCs differentiation and interact with the RNA-binding protein ELAVL1/HuR. Furthermore, we characterize the function of two of the deregulated lncRNAs, lincBRN1a and lincBRN1b, during NSCs' differentiation. Their inhibition leads to the induction of differentiation, with a concomitant decrease in stemness and an increase in neuronal markers, indicating that they exert key functions in neuronal cells differentiation. Furthermore, we describe here that HuR regulates their half-life, suggesting their synergic role in the differentiation process. We also identify six human homologs (PANTR1, TUG1, HOTTIP, TP53COR, ELDRR and FENDRR) of the mentioned 10 lncRNAs and we report their deregulation during human iPSCs differentiation into neurons. In conclusion, our results strongly indicate a key synergic role for lncRNAs and HuR in neuronal stem cells fate.
Subject(s)
Cell Differentiation/genetics , ELAV-Like Protein 1/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , RNA, Long Noncoding/genetics , Animals , Biomarkers , Cell Self Renewal/genetics , Gene Expression Regulation, Developmental , Gene Silencing , Humans , Immunohistochemistry , Male , MiceABSTRACT
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.
Subject(s)
ELAV-Like Protein 1/chemistry , Phenanthrenes/chemistry , Phenanthrenes/pharmacology , 3' Untranslated Regions , AU Rich Elements , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , ELAV-Like Protein 1/antagonists & inhibitors , ELAV-Like Protein 1/genetics , ELAV-Like Protein 1/metabolism , Furans , Humans , Magnetic Resonance Spectroscopy , Mice, Neurologic Mutants , Molecular Dynamics Simulation , Phenanthrenes/metabolism , Point Mutation , Protein Conformation , Protein Domains , Quinones , RNA, Messenger/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Obesity and insulin resistance are associated with oxidative stress, which may be implicated in the progression of obesity-related diseases. The kinase JNK1 has emerged as a promising drug target for the treatment of obesity and type 2 diabetes. JNK1 is also a key mediator of the oxidative stress response, which can promote cell death or survival, depending on the magnitude and context of its activation. In this article, we describe a study in which the long-term effects of JNK1 inactivation on glucose homeostasis and oxidative stress in obese mice were investigated for the first time. Mice lacking JNK1 (JNK1(-/-)) were fed an obesogenic high-fat diet (HFD) for a long period. JNK1(-/-) mice fed an HFD for the long term had reduced expression of antioxidant genes in their skin, more skin oxidative damage, and increased epidermal thickness and inflammation compared with the effects in control wild-type mice. However, we also observed that the protection from obesity, adipose tissue inflammation, steatosis, and insulin resistance, conferred by JNK1 ablation, was sustained over a long period and was paralleled by decreased oxidative damage in fat and liver. We conclude that compounds targeting JNK1 activity in brain and adipose tissue, which do not accumulate in the skin, may be safer and most effective.-Becattini, B., Zani, F., Breasson, L., Sardi, C., D'Agostino, V. G., Choo, M.-K., Provenzani, A., Park, J. M., Solinas, G. JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage.
Subject(s)
Dietary Fats/adverse effects , Mitogen-Activated Protein Kinase 8/metabolism , Obesity/chemically induced , Skin/pathology , Aging , Animals , Dietary Fats/administration & dosage , Fatty Liver/chemically induced , Fatty Liver/metabolism , Inflammation/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 8/genetics , Oxidative StressABSTRACT
Since 2005, sex determining region y-box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-α mRNA. Our results show that HuR binds to the 3'-untranslated region of SOX2 mRNA together with the RNA-induced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 3'-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay.
Subject(s)
ELAV-Like Protein 1/metabolism , Mesenchymal Stem Cells/metabolism , RNA, Messenger/physiology , SOXB1 Transcription Factors/physiology , Cells, Cultured , Female , Humans , Male , Protein Binding/physiologyABSTRACT
Boosting NAD(+) biosynthesis with NAD(+) intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD(+) biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFß signaling pathway via increased TGFß1 production. These findings have implications for the design of therapeutic strategies exploiting NAD(+) biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.
Subject(s)
Breast Neoplasms/genetics , Cytokines/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/genetics , Nicotinamide Phosphoribosyltransferase/genetics , Transforming Growth Factor beta1/genetics , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Cytokines/antagonists & inhibitors , Cytokines/metabolism , Female , Humans , NAD/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Neoplasm Staging , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Nicotinamide Phosphoribosyltransferase/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Receptors, Estrogen/deficiency , Receptors, Estrogen/genetics , Signal Transduction , Transforming Growth Factor beta1/metabolismABSTRACT
BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide, is one of the major factors regulating cancer cells metabolism and is considered a promising target for treating cancer. The prototypical NAMPT inhibitor FK866 effectively lowers NAD(+) levels in cancer cells, reducing the activity of NAD(+)-dependent enzymes, lowering intracellular ATP, and promoting cell death. RESULTS: We show that FK866 induces a translational arrest in leukemia cells through inhibition of MTOR/4EBP1 signaling and of the initiation factors EIF4E and EIF2A. Specifically, treatment with FK866 is shown to induce 5'AMP-activated protein kinase (AMPK) activation, which, together with EIF2A phosphorylation, is responsible for the inhibition of protein synthesis. Notably, such an effect was also observed in patients' derived primary leukemia cells including T-cell Acute Lymphoblastic Leukemia. Jurkat cells in which AMPK or LKB1 expression was silenced or in which a non-phosphorylatable EIF2A mutant was ectopically expressed showed enhanced sensitivity to the NAMPT inhibitor, confirming a key role for the LKB1-AMPK-EIF2A axis in cell fate determination in response to energetic stress via NAD(+) depletion. CONCLUSIONS: We identified EIF2A phosphorylation as a novel early molecular event occurring in response to NAMPT inhibition and mediating protein synthesis arrest. In addition, our data suggest that tumors exhibiting an impaired LBK1- AMPK- EIF2A response may be especially susceptible to NAMPT inhibitors and thus become an elective indication for this type of agents.
Subject(s)
Cytokines/antagonists & inhibitors , Eukaryotic Initiation Factor-2/metabolism , Leukemia/genetics , Leukemia/metabolism , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Protein Biosynthesis , Stress, Physiological/drug effects , AMP-Activated Protein Kinases/metabolism , Acrylamides/pharmacology , Adenosine Triphosphate/metabolism , Apoptosis/drug effects , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm , Eukaryotic Initiation Factor-4E/metabolism , Humans , Jurkat Cells , NAD/metabolism , Phosphorylation , Piperidines/pharmacology , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Transcription, GeneticABSTRACT
Little is known regarding the post-transcriptional networks that control gene expression in eukaryotes. Additionally, we still need to understand how these networks evolve, and the relative role played in them by their sequence-dependent regulatory factors, non-coding RNAs (ncRNAs) and RNA-binding proteins (RBPs). Here, we used an approach that relied on both phylogenetic sequence sharing and conservation in the whole mapped 3'-untranslated regions (3'-UTRs) of vertebrate species to gain knowledge on core post-transcriptional networks. The identified human hyper conserved elements (HCEs) were predicted to be preferred binding sites for RBPs and not for ncRNAs, namely microRNAs and long ncRNAs. We found that the HCE map identified a well-known network that post-transcriptionally regulates histone mRNAs. We were then able to discover and experimentally confirm a translational network composed of RNA Recognition Motif (RRM)-type RBP mRNAs that are positively controlled by HuR, another RRM-type RBP. HuR shows a preference for these RBP mRNAs bound in stem-loop motifs, confirming its role as a 'regulator of regulators'. Analysis of the transcriptome-wide HCE distribution revealed a profile of prevalently small clusters separated by unconserved intercluster RNA stretches, which predicts the formation of discrete small ribonucleoprotein complexes in the 3'-UTRs.
Subject(s)
3' Untranslated Regions , ELAV Proteins/physiology , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Animals , Base Sequence , Binding Sites , Conserved Sequence , ELAV Proteins/genetics , ELAV Proteins/metabolism , Histones/genetics , Humans , Inverted Repeat Sequences , MCF-7 Cells , Protein Biosynthesis , Sequence Alignment , VertebratesABSTRACT
The protein kinase Cδ (PKCδ) interacts with and phosphorylates HuR, dictating its functionality. We show here that the genotoxic stimulus induced by doxorubicin triggers PKCδ interaction with HuR and leads to HuR phosphorylation on serines 221 and 318 and cytoplasmic translocation. This series of events is crucial to elicit the death pathway triggered by doxorubicin and is necessary to promote HuR function in post-transcriptional regulation of gene expression, because genetic ablation of PKCδ caused the inability of HuR to bind its target mRNAs, topoisomerase IIα (TOP2A) included. In in vitro select doxorubicin-resistant human breast cancer cell lines upregulating the multidrug resistance marker ABCG2, PKCδ, and HuR proteins were coordinately downregulated together with the doxorubicin target TOP2A protein whose mRNA was HuR-regulated. Therefore, we show here that PKCδ, HuR, and TOP2A constitute a network mediating doxorubicin efficacy in breast cancer cells. The importance of these molecular events in cancer therapy is suggested by their being profoundly suppressed in cells selected for doxorubicin resistance.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Breast Neoplasms/metabolism , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , ELAV Proteins/metabolism , Protein Kinase C-delta/metabolism , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation , ELAV Proteins/genetics , Female , Humans , Phosphorylation , Poly-ADP-Ribose Binding Proteins , Protein Kinase C-delta/genetics , RNA, Small Interfering/geneticsABSTRACT
RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein-protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized.
Subject(s)
Biotin/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group C/chemistry , Protein Interaction Mapping , Proteome/analysis , Amino Acid Sequence , Biological Assay , Carbon-Nitrogen Ligases/chemistry , Carbon-Nitrogen Ligases/genetics , Carbon-Nitrogen Ligases/metabolism , ELAV Proteins/chemistry , ELAV Proteins/genetics , ELAV Proteins/metabolism , ELAV-Like Protein 1 , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Heterogeneous-Nuclear Ribonucleoprotein Group C/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group C/metabolism , Humans , Molecular Sequence Data , Nuclear Matrix-Associated Proteins/chemistry , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , Poly(A)-Binding Protein I/chemistry , Poly(A)-Binding Protein I/genetics , Poly(A)-Binding Protein I/metabolism , Protein Binding , Protein Biosynthesis , Protein Interaction Maps , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/metabolism , Streptavidin/chemistryABSTRACT
The life of an mRNA molecule begins with transcription and ultimately ends in degradation. In the course of its life, however, mRNA is examined, modified in various ways and transported before eventually being translated into proteins. All these processes are performed by proteins and non-coding RNAs whose complex interplay in the cell contributes to determining the proteome changes and the phenotype of cells. On May 23â26, 2012, over 150 scientists from around the world convened in the sunny shores of Riva del Garda, Italy, for the workshop entitled: "mRNA fate: Life and Death of mRNA in the Cytoplasm." Sessions included mRNA trafficking, mRNA translational control, RNA metabolism and disease, RNA-protein structures and systems biology of RNA. This report highlights some of the prominent and recurring themes at the meeting and emerging arenas of future research.
Subject(s)
Cytoplasm/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Animals , Gene Expression Regulation , Humans , MicroRNAs/chemistry , MicroRNAs/genetics , MicroRNAs/metabolism , Phenotype , Protein Biosynthesis , Proteins/chemistry , Proteins/metabolism , Proteome , RNA Stability , RNA, Messenger/chemistryABSTRACT
Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5ß interacts with the scaffold ERC1, and recruits it at plasma membrane-associated platforms that form at the front of migrating tumor cells. LL5 and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5ß and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5ß(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5ß protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5ß(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5ß interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5ß. Expression of LL5ß(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane-associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion.
Subject(s)
Cell Membrane , Cell Movement , Immunoprecipitation , MDA-MB-231 Cells , HumansABSTRACT
Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD+ synthesis pathways and is found upregulated in several tumors, depicting NAD(H) lowering agents, like the NAMPT inhibitor FK866, as an appealing approach for anticancer therapy. Like other small molecules, FK866 triggers chemoresistance, observed in several cancer cellular models, which can prevent its clinical application. The molecular mechanisms sustaining the acquired of resistance to FK866 were studied in a model of triple negative breast cancer (MDA-MB-231 parental - PAR), exposed to increasing concentrations of the small molecule (MDA-MB-231 resistant - RES). RES cells are not sensitive to verapamil or cyclosporin A, excluding a potential role of increased efflux pumps activity as a mechanism of resistance. Similarly, the silencing of the enzyme Nicotinamide Riboside Kinase 1 (NMRK1) in RES cells does not increase FK866 toxicity, excluding this pathway as a compensatory mechanism of NAD+ production. Instead, Seahorse metabolic analysis revealed an increased mitochondrial spare respiratory capacity in RES cells. These cells presented a higher mitochondrial mass compared to the FK866-sensitive counterparts, as well as an increased consumption of pyruvate and succinate for energy production. Interestingly, co-treatment of PAR cells with FK866 and the mitochondrial pyruvate carrier (MPC) inhibitors UK5099 or rosiglitazone, as well as with the transient silencing of MPC2 but not of MPC1, induces a FK866-resistant phenotype. Taken together, these results unravel novel mechanisms of cell plasticity to counteract FK866 toxicity, that, besides the previously described LDHA dependency, rely on mitochondrial rewiring at functional and energetic levels.
Subject(s)
NAD , Triple Negative Breast Neoplasms , Humans , NAD/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Cytokines/metabolism , Nicotinamide Phosphoribosyltransferase/genetics , Nicotinamide Phosphoribosyltransferase/metabolism , Mitochondria/metabolism , Cell Line, Tumor , Phosphotransferases (Alcohol Group Acceptor)ABSTRACT
Lipopolysaccharide (LPS) exposure to macrophages induces an inflammatory response, which is regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA-binding protein that regulates cytokines and chemokines transcripts containing AU/U-rich elements (AREs) and mediates the LPS-induced response. Here, we show that small-molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. TMs exist in solution in keto-enolic tautomerism, and molecular dynamic calculations showed the ortho-quinone form inhibiting binding of HuR to mRNA targets. TM activity was lost in vitro by blocking the diphenolic reduced form as a diacetate, but resulted in prodrug-like activity in vivo. RNA and ribonucleoprotein immunoprecipitation sequencing revealed that LPS induces a strong coupling between differentially expressed genes and HuR-bound genes, and TMs reduced such interactions. TMs decreased the association of HuR with genes involved in chemotaxis and immune response, including Cxcl10, Il1b and Cd40, reducing their expression and protein secretion in primary murine bone marrow-derived macrophages and in an LPS-induced peritonitis model. Overall, TMs show anti-inflammatory properties in vivo and suggest HuR as a potential therapeutic target for inflammation-related diseases.
Subject(s)
ELAV-Like Protein 1 , Lipopolysaccharides , Mice , Animals , Lipopolysaccharides/pharmacology , Lipopolysaccharides/metabolism , ELAV-Like Protein 1/genetics , ELAV-Like Protein 1/metabolism , Macrophages/metabolism , RNA/metabolism , RNA, Messenger/geneticsABSTRACT
BACKGROUND: HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7). RESULTS: We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. Doxorubicin-induced apoptosis was also correlated with the presence of HuR in the cytoplasm. Rottlerin, which was able to block HuR nuclear export, had correspondingly antagonistic effects with doxorubicin on cell toxicity. The proapoptotic activity of HuR was not due to cleavage to an active form, as was previously reported. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response. CONCLUSIONS: The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin and that restoration of HuR expression in resistant cells resensitizes them to the action of this drug, thereby identifying HuR as a key protein in doxorubicin pharmacology.
Subject(s)
Breast Neoplasms/metabolism , Doxorubicin/pharmacology , ELAV Proteins/metabolism , Acetophenones/pharmacology , Apoptosis/drug effects , Benzopyrans/pharmacology , Biological Transport/drug effects , Cell Line, Tumor , Drug Resistance, Neoplasm/physiology , ELAV Proteins/genetics , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Immunoprecipitation , Oligonucleotide Array Sequence Analysis , Phosphorylation/drug effectsABSTRACT
Germline CDKN2A mutations are observed in 20-50% of melanoma-prone families. We identified melanoma patients that were heterozygous for non-coding germline variants in the 5'-UTR of CDKN2A (c.-21C > T; c.-25C > T&c.-180G > A; c.-56G > T; c.-67G > C) and examined their impact on the p16(INK4a) 5'-UTR activity using two luciferase-based reporter vectors that differ in basal transcription level and that were transfected into the melanoma-derived WM266-4 and in the breast cancer-derived MCF7 cells. The wild-type 5'-UTR sequence, containing a reported SNP (c.-33G > C) and a known melanoma-predisposing mutation (c.-34G > T), was included as controls. Results revealed that the variants at -21 and -34 severely reduced the reporter activity. The variants at -56 and at -25&-180 exhibited a milder impact, while results with c.-67G > C were dependent on the plasmid type. Quantification of the luciferase mRNA indicated that the effects of the variants were mainly post-transcriptional. Using a bicistronic dual-luciferase reporter plasmid, we confirmed that c.-21C > T and c.-34G > T had a severe negative impact in both cell lines. We also applied a polysomal profiling technique to samples heterozygous for the 5'-UTR variants, including patient-derived lymphoblasts. Analysis of allelic imbalance indicated that in addition to the c.-21C > T variant, the c.-56T > G and c.-67G > C variants also reduced mRNA translation efficiency. Overall, our results suggest that the c.-21C > T sequence variant is a melanoma-predisposing mutation. The c.-25C > T&c.-180G > A and particularly the c.-56G > T variants showed a range of intermediate functional defects in the different assays, and were not observed in the control population. We propose that these variants should be considered as potential mutations.
Subject(s)
5' Untranslated Regions , Cyclin-Dependent Kinase Inhibitor p16/genetics , Genetic Predisposition to Disease , Genetic Variation , Melanoma/genetics , Cell Line, Tumor , Cohort Studies , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Female , Humans , Italy , Male , Melanoma/metabolism , Mutation , Pedigree , Polymorphism, Single NucleotideABSTRACT
Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical ß1α1ß2ß3α2ß4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.