RESUMO
Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Neoplasias Pulmonares/genética , Proliferação de Células/genética , Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
BACKGROUND: Neuroendocrine tumors (NETs) overexpress somatostatin receptors (SSTRs). METHODS: We developed a second-generation, ligand-based, anti-SSTR chimeric antigen receptor (CAR) incorporating the somatostatin analog octreotide in its extracellular moiety. RESULTS: Anti-SSTR CAR T cells exerted antitumor activity against SSTR+NET cell linesin vitro. The killing activity was highly specific, as demonstrated by the lack of CAR T cell reactivity against NET cells engineered to express mutated variants of SSTR2/5 by CRISPR/Cas9. When adoptively transferred in NSG mice, anti-SSTR CAR T cells induced significant antitumor activity against human NET xenografts. Although anti-SSTR CAR T cells could recognize the murine SSTRs as shown by their killing ability against murine NET cells, no obvious deleterious effects on SSTR-expressing organs such as the brain or the pancreas were observed in mice. CONCLUSIONS: Taken together, our results establish anti-SSTR CAR T cells as a potential candidate for early phase clinical investigations in patients with NETs. More broadly, the demonstration that a known peptide drug can direct CAR T cell targeting may streamline the potential utility of multiple peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Tumores Neuroendócrinos , Animais , Humanos , Ligantes , Camundongos , Tumores Neuroendócrinos/tratamento farmacológico , Octreotida , Somatostatina/uso terapêuticoRESUMO
The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation.
Assuntos
Bactérias/crescimento & desenvolvimento , Meios de Cultivo Condicionados/química , Drosophila/crescimento & desenvolvimento , Vesículas Extracelulares/metabolismo , Fungos/crescimento & desenvolvimento , Neoplasias/metabolismo , Animais , Bactérias/química , Células CACO-2 , Estudos de Casos e Controles , Drosophila/química , Difusão Dinâmica da Luz , Citometria de Fluxo , Fungos/química , Voluntários Saudáveis , Humanos , Nanopartículas , Tamanho da Partícula , UltracentrifugaçãoRESUMO
Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.
Assuntos
Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster , Doenças Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Trifosfato de Adenosina/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Atividade Motora/fisiologia , Mutação , Fenótipo , Reprodução/fisiologiaRESUMO
We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1 We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML.
Assuntos
Diferenciação Celular/genética , Expressão Ectópica do Gene , Epigênese Genética , Proteínas de Homeodomínio/genética , Células Mieloides/citologia , Células Mieloides/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional/métodos , DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , DNA Metiltransferase 3A , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Estudos de Associação Genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Mutação , Nucleofosmina , Translocação Genética , Adulto JovemRESUMO
The transposons of the Bari family are mobile genetic elements widespread in the Drosophila genus. However, despite a broad diffusion, virtually no information is available on the mechanisms underlying their mobility. In this paper we report the functional characterization of the Bari elements transposition system. Using the Bari1 element as a model, we investigated the subcellular localization of the transposase, its physical interaction with the transposon, and its catalytic activity. The Bari1 transposase localized in the nucleus and interacted with the terminal sequences of the transposon both in vitro and in vivo, however, no transposition activity was detected in transposition assays. Profiling of mRNAs expressed by the transposase gene revealed the expression of abnormal, internally processed transposase transcripts encoding truncated, catalytically inactive transposase polypeptides. We hypothesize that a post-transcriptional control mechanism produces transposase-derived polypeptides that effectively repress transposition. Our findings suggest further clues towards understanding the mechanisms that control transposition of an important class of mobile elements, which are both an endogenous source of genomic variability and widely used as transformation vectors/biotechnological tools.
Assuntos
Elementos de DNA Transponíveis , Drosophila/genética , Drosophila/metabolismo , Transposases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Choque Térmico/genética , Humanos , Espaço Intracelular/metabolismo , Sequências Repetidas Invertidas , Masculino , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Transporte Proteico , Splicing de RNA , Alinhamento de Sequência , Transcrição Gênica , Transposases/química , Transposases/genéticaRESUMO
The exact mechanism by which ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling is not known. ENPP1 contains two somatomedin-B-like domains (i.e. SMB 1 and 2) involved in ENPP1 dimerization in animal cells. The aim of the present study was to investigate if these domains modulate ENPP1 inhibitory activity on insulin signaling in human insulin target cells (HepG2). ENPP1 (ENPP1-3'myc), ENPP1 deleted of SMB 1 (ENPP1-ΔI-3'myc) or of SMB 2 (ENPP1-ΔII-3'myc) domain were cloned in frame with myc tag in mammalian expression vector pRK5. Plasmids were transiently transfected in human liver HepG2 cells. ENPP1 inhibitory activity on insulin signaling, dimerization and protein-protein interaction with insulin receptor (IR), reported to mediate the modulation of ENPP1 inhibitory activity, were studied. As compared to untransfected cells, a progressive increase of ENPP1 inhibitory activity on insulin-induced IR ß-subunit autophosphorylation and on Akt-S(473) phosphorylation was observed in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. Under non reducing conditions a 260 kDa homodimer, indicating ENPP1 dimerization, was observed. The ratio of non reduced (260 kDa) to reduced (130 kDa) ENPP1 was significantly decreased by two thirds in ENPP1-ΔII-3'myc vs. ENPP1-3'myc but not in ENPP1-ΔI-3'myc. A similar ENPP1/IR interaction was detectable by co-immunoprecipitation in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. In conclusion, SMB 1 and SMB 2 are negative modulators of ENPP1 inhibitory activity on insulin signaling. For SMB 2 such effect might be mediated by a positive role on protein dimerization.
Assuntos
Insulina/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Somatomedinas/metabolismo , Western Blotting , Células Hep G2 , Humanos , Imunoprecipitação , Insulina/química , Diester Fosfórico Hidrolases/genética , Fosforilação , Plasmídeos , Multimerização Proteica , Estrutura Terciária de Proteína , Pirofosfatases/genéticaRESUMO
Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)-encoded RNAs and nuclear DNA-encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis.