RESUMO
Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.
Assuntos
Transferases Intramoleculares/metabolismo , Biossíntese de Proteínas , Pseudouridina/metabolismo , RNA de Transferência/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Fatores de Iniciação em Eucariotos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Transferases Intramoleculares/antagonistas & inibidores , Transferases Intramoleculares/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Síndromes Mielodisplásicas/patologia , Conformação de Ácido Nucleico , Fosfoproteínas/metabolismo , Proteína I de Ligação a Poli(A)/antagonistas & inibidores , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Nicho de Células-TroncoRESUMO
SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.
Assuntos
Leucemia , Síndromes Mielodisplásicas , Fosfoproteínas , Fatores de Processamento de RNA , Animais , Humanos , Camundongos , Carcinogênese/genética , Leucemia/genética , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceosomal components are co-opted during tumorigenesis remains poorly defined. Here we unravel the core splice factor SF3A3 at the nexus of a translation-based program that rewires splicing during malignant transformation. Upon MYC hyperactivation, SF3A3 levels are modulated translationally through an RNA stem-loop in an eIF3D-dependent manner. This ensures accurate splicing of mRNAs enriched for mitochondrial regulators. Altered SF3A3 translation leads to metabolic reprogramming and stem-like properties that fuel MYC tumorigenic potential in vivo. Our analysis reveals that SF3A3 protein levels predict molecular and phenotypic features of aggressive human breast cancers. These findings unveil a post-transcriptional interplay between splicing and translation that governs critical facets of MYC-driven oncogenesis.
Assuntos
Neoplasias da Mama/metabolismo , Carcinogênese/metabolismo , Células-Tronco Neoplásicas/metabolismo , Biossíntese de Proteínas , Fatores de Processamento de RNA/biossíntese , Spliceossomos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Processamento de RNA/genética , Spliceossomos/genéticaRESUMO
Benbarche, Pineda, Galvis, et al. delineate an essential role for the G-patch motif-containing protein GPATCH8 in mis-splicing associated with cancer-driving mutations of the splicing factor SF3B1. GPATCH8 cooperates with SF3B1 mutants, affecting the splicing machinery. Targeting GPATCH8 reveals therapeutic opportunities for SF3B1 mutant cancers and other splicing-related diseases.
Assuntos
Neoplasias , Fatores de Processamento de RNA , Splicing de RNA , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/genéticaRESUMO
Perturbation of protein phosphorylation represents an attractive approach to cancer treatment. Besides kinase inhibitors, protein phosphatase inhibitors have been shown to have anti-cancer activity. A prime example is the small molecule LB-100, an inhibitor of protein phosphatases 2A/5 (PP2A/PP5), enzymes that affect cellular physiology. LB-100 has proven effective in pre-clinical models in combination with immunotherapy, but the molecular underpinnings of this synergy remain understood poorly. We report here a sensitivity of the mRNA splicing machinery to phosphorylation changes in response to LB-100 in colorectal adenocarcinoma. We observe enrichment for differentially phosphorylated sites within cancer-critical splicing nodes of U2 snRNP, SRSF and hnRNP proteins. Altered phosphorylation endows LB-100-treated colorectal adenocarcinoma cells with differential splicing patterns. In PP2A-inhibited cells, over 1000 events of exon skipping and intron retention affect regulators of genomic integrity. Finally, we show that LB-100-evoked alternative splicing leads to neoantigens that are presented by MHC class 1 at the cell surface. Our findings provide a potential explanation for the pre-clinical and clinical observations that LB-100 sensitizes cancer cells to immune checkpoint blockade.
Assuntos
Neoplasias do Colo , Splicing de RNA , Humanos , Processamento Alternativo/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Inibidores Enzimáticos/farmacologia , Fosforilação , Proteína Fosfatase 2/metabolismo , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Piperazinas/farmacologiaRESUMO
While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1-/- ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1-/- mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1-/- animals have reduced quiescence and regenerative potential. Young HO-1-/- HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1+/+ HSCs transplanted into HO-1-/- recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1-/- HSCs to the HO-1+/+ recipients recovers the regenerative potential of HO-1-/- HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.
Assuntos
Heme Oxigenase-1 , Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Células Endoteliais , Células-Tronco Hematopoéticas , Heme Oxigenase-1/genéticaRESUMO
Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.
Assuntos
Heme Oxigenase-1 , Melanoma Experimental , Animais , Linhagem Celular Tumoral , Separação Celular , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Melanoma Experimental/patologia , Proteínas de Membrana , Camundongos , Células-Tronco Neoplásicas/metabolismoRESUMO
Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(-413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients.
Assuntos
Neutropenia Febril Induzida por Quimioterapia/genética , Resistencia a Medicamentos Antineoplásicos/genética , Heme Oxigenase-1/genética , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Cultivadas , Neutropenia Febril Induzida por Quimioterapia/etiologia , Criança , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Regiões Promotoras GenéticasRESUMO
Heme oxygenase-1 (HO-1, Hmox1) regulates viability, proliferation, and differentiation of many cell types; hence, it may affect regeneration of injured skeletal muscle. Here, we injected cardiotoxin into gastrocnemius muscle of Hmox1+/+ and Hmox1-/- animals and analyzed cellular response after muscle injury, focusing on muscle satellite cells (SCs), inflammatory reaction, fibrosis, and formation of new blood vessels. HO-1 is strongly induced after muscle injury, being expressed mostly in the infiltrating leukocytes (CD45+ cells), including macrophages (F4/80+ cells). Lack of HO-1 augments skeletal muscle injury, evidenced by increased creatinine kinase and lactate dehydrogenase, as well as expression of monocyte chemoattractant protein-1, IL-6, IL-1ß, and insulin-like growth factor-1. This, together with disturbed proportion of M1/M2 macrophages, accompanied by enhanced formation of arterioles, may be responsible for shift of Hmox1-/- myofiber size distribution toward larger one. Importantly, HO-1-deficient SCs are prone to activation and have higher proliferation on injury. This effect can be partially mimicked by stimulation of Hmox1+/+ SCs with monocyte chemoattractant protein-1, IL-6, IL-1ß, and is associated with increased MyoD expression, suggesting that Hmox1-/- SCs are shifted toward more differentiated myogenic population. However, multiple rounds of degeneration/regeneration in conditions of HO-1 deficiency may lead to exhaustion of SC pool, and the number of SCs is decreased in old Hmox1-/- mice. In summary, HO-1 modulates muscle repair mechanisms preventing its uncontrolled acceleration.
Assuntos
Heme Oxigenase-1/fisiologia , Músculo Esquelético/lesões , Miosite/enzimologia , Células Satélites de Músculo Esquelético/patologia , Animais , Arteríolas/patologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Proteínas Cardiotóxicas de Elapídeos , Crotoxina , Citocinas/biossíntese , Combinação de Medicamentos , Feminino , Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/deficiência , Heme Oxigenase-1/genética , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miosite/induzido quimicamente , Miosite/patologia , Miosite/fisiopatologia , RNA Mensageiro/genética , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/metabolismoRESUMO
Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1-/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2-/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018.
Assuntos
Diferenciação Celular/fisiologia , Técnicas de Reprogramação Celular/métodos , Heme Oxigenase-1/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Membrana/metabolismo , Animais , Ciclo Celular/fisiologia , Fibroblastos , Heme Oxigenase-1/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
AIMS: Heme oxygenase-1 (HO-1; HMOX1 in human, Hmox1 in mice) is an antioxidative enzyme affecting wide range of sub-cellular processes. It was shown to modulate tumor growth or vascular-related diseases, thus being putative molecular target for tailored therapies. Therefore it is of importance to elucidate novel compounds regulating HO-1 activity/expression and to delineate mechanisms of their action. In the present study we aimed to understand mode of action of valproic acid (VA), an antiepileptic drug, on HO-1 expression. RESULTS: We demonstrated that HO-1 expression is decreased by VA at protein but not mRNA level in human alveolar rhabdomyosarcoma cell line CW9019. Nrf2 transcription factor, the activator of HO-1 expression through ARE sequence, was excluded as a mediator of HO-1 decrease, as VA downregulated Bach1, a Nrf2 repressor, concomitantly upregulating ARE activation. Also miRNA-dependent inhibition was excluded as a mechanism of HMOX1 regulation. However, co-immunoprecipitation assay showed a higher level of ubiquitinated HO-1 after VA treatment. Accordingly, MG132, an inhibitor of proteasomal degradation, reversed the effect of VA on HO-1 suggesting that decrease in HO-1 expression by VA is through protein stability. The inhibitory effect of VA on HO-1 was also observed in murine cells including embryonic fibroblasts isolated from Nrf2-deficient mice, what confirms Nrf2-independent effect of the compound. Importantly, VA decreased also HO-1 expression and activity in murine skeletal muscles in vivo. CONCLUSION: Our data indicate that VA downregulates HO-1 by acting through ubiquitin-proteasomal pathway leading to decrease in protein level.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Ácido Valproico/farmacologia , Animais , Anticonvulsivantes/farmacologia , Linhagem Celular , Heme Oxigenase-1/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Huntington's disease (HD) is a devastating, neurodegenerative condition, which lacks effective treatment. Normal Huntingtin (HTT) and mutant Huntingtin (mHTT) are expressed in multiple tissues and can alter transcription of microRNAs (miRs). Importantly, miRs are present in a bio-stable form in human peripheral blood plasma and have recently been shown to be useful biomarkers in other diseases. We therefore sought to identify potential miR biomarkers of HD that are present in, and have functional consequences for, neuronal and non-neuronal tissues. In a cell line over-expressing mHTT-Exon-1, miR microarray analysis was used to identify candidate miRs. We then examined their presence and bio-stability in control and HD plasma. We found that miR-34b is significantly elevated in response to mHTT-Exon-1, and its blockade alters the toxicity of mHTT-Exon-1 in vitro. We also show that miR-34b is detectable in plasma from small input volumes and is insensitive to freeze-thaw-induced RNA degradation. Interestingly, miR-34b is significantly elevated in plasma from HD gene carriers prior to symptom onset. This is the first study suggesting that plasma miRs might be used as biomarkers for HD.
Assuntos
Doença de Huntington/genética , MicroRNAs/sangue , Adulto , Idoso , Biomarcadores/sangue , Morte Celular , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Éxons , Feminino , Humanos , Doença de Huntington/sangue , Imuno-Histoquímica , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Mutação , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transfecção , Regulação para Cima , Adulto JovemRESUMO
Diabetes has been detected in Danish and Swedish bank voles (Myodes glareolus). There are no data, however, concerning the prevalence of diabetes in populations from other geographic regions. We investigated the frequency and physiological effects of glucose metabolism disorders in captive bank voles from Poland. Single measurement of fasting blood glucose concentration performed in the 3-4month old captive-born bank Polish voles without any disease symptoms showed that 8% of individuals (22/284) displayed an impaired fasting glucose (IFG, blood glucose (BG) ≥100mg/dL) and 1% (4/284) showed hyperglycemia (BG ≥126mg/dL) which could suggest diabetes. Next, we analyzed blood glucose in samples taken once a month from an additional cohort of bank voles with (FHD), or without (H), a family history of diabetes. The prevalence of IFG at age six months was 26% (16/62) among bank voles from the H group. In the FHD group the prevalence increased to 49% (43/88), and additional 12% (11/88) became diabetic (DB, BG ≥126mg/dL at two time points). Postnatal stress (three maternal deprivations before weaning) did not affect the risk of developing IFG or DB in H voles, but significantly reduced the frequency of glucose metabolism disorders (IFG and DB combined) in FHD voles. IFG was associated with hyperinsulinemia, but not with other biochemical disturbances. Diabetic animals displayed a progressive malformation and vacuolization of ß-cells in the pancreas, without visible leukocytic infiltrations. In summary, our results indicate that Polish captive bank voles can develop diabetes, which shows features of both type 1 and type 2 diabetes in humans. Risk of diabetes is higher in animal with FHD.
Assuntos
Arvicolinae/metabolismo , Diabetes Mellitus/veterinária , Glucose/metabolismo , Hiperglicemia/veterinária , Doenças dos Roedores/metabolismo , Animais , Glicemia , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/metabolismo , Hiperglicemia/epidemiologia , Hiperglicemia/metabolismo , Privação Materna , Prevalência , Fatores de Risco , Doenças dos Roedores/epidemiologia , Estresse FisiológicoRESUMO
The LIN28B RNA binding protein exhibits an ontogenically restricted expression pattern and is a key molecular regulator of fetal and neonatal B lymphopoiesis. It enhances the positive selection of CD5+ immature B cells early in life through amplifying the CD19/PI3K/c-MYC pathway and is sufficient to reinitiate self-reactive B-1a cell output when ectopically expressed in the adult. In this study, interactome analysis in primary B cell precursors showed direct binding by LIN28B to numerous ribosomal protein transcripts, consistent with a regulatory role in cellular protein synthesis. Induction of LIN28B expression in the adult setting is sufficient to promote enhanced protein synthesis during the small Pre-B and immature B cell stages, but not during the Pro-B cell stage. This stage dependent effect was dictated by IL-7 mediated signaling, which masked the impact of LIN28B through an overpowering stimulation on the c-MYC/protein synthesis axis in Pro-B cells. Importantly, elevated protein synthesis was a distinguishing feature between neonatal and adult B cell development that was critically supported by endogenous Lin28b expression early in life. Finally, we used a ribosomal hypomorphic mouse model to demonstrate that subdued protein synthesis is specifically detrimental for neonatal B lymphopoiesis and the output of B-1a cells, without affecting B cell development in the adult. Taken together, we identify elevated protein synthesis as a defining requirement for early-life B cell development that critically depends on Lin28b. Our findings offer new mechanistic insights into the layered formation of the complex adult B cell repertoire.
Assuntos
Linfócitos B , Células Precursoras de Linfócitos B , Camundongos , AnimaisRESUMO
Slow-cycling cancer cells (SCC) contribute to the aggressiveness of many cancers, and their invasiveness and chemoresistance pose a great therapeutic challenge. However, in melanoma, their tumor-initiating abilities are not fully understood. In this study, we used the syngeneic transplantation assay to investigate the tumor-initiating properties of melanoma SCC in the physiologically relevant in vivo settings. For this we used B16-F10 murine melanoma cell line where we identified a small fraction of SCC. We found that, unlike human melanoma, the murine melanoma SCC were not marked by the high expression of the epigenetic enzyme Jarid1b. At the same time, their slow-cycling phenotype was a temporary state, similar to what was described in human melanoma. Progeny of SCC had slightly increased doxorubicin resistance and altered expression of melanogenesis genes, independent of the expression of cancer stem cell markers. Single-cell expansion of SCC revealed delayed growth and reduced clone formation when compared to non-SCC, which was further confirmed by an in vitro limiting dilution assay. Finally, syngeneic transplantation of 10 cells in vivo established that SCC were able to initiate growth in primary recipients and continue growth in the serial transplantation assay, suggesting their self-renewal nature. Together, our study highlights the high plasticity and tumorigenicity of murine melanoma SCC and suggests their role in melanoma aggressiveness.
Assuntos
Melanoma Experimental , Humanos , Animais , Camundongos , Transplante Isogênico , Melanoma Experimental/genética , Melanoma Experimental/tratamento farmacológico , Linhagem Celular , Proliferação de CélulasRESUMO
To realize the potential of microRNAs (miRs) as fine-tuning regulators of embryonic neuronal differentiation, it is critical to define their developmental function. Mmu-miR-134 (miR-134) is a powerful inducer of pluripotent stem cell differentiation. However, its functional role during embryonic, neuronal development is unknown. We demonstrate that mature, miR-134 transcript levels elevate during embryonic, neuronal differentiation in vitro and in vivo. To define the developmental targets and function of miR-134, we identified multiple brain-expressed targets including the neural progenitor cell-enriched, bone morphogenetic protein (BMP) antagonist Chordin-like 1 (Chrdl-1) and the postmitotic, neuron-specific, microtubule-associated protein, Doublecortin (Dcx). We show that, through interaction with Dcx and/or Chrdl-1, miR-134 has stage-specific effects on cortical progenitors, migratory neurons, and differentiated neurons. In neural progenitors, miR-134 promotes cell proliferation and counteracts Chrdl-1-induced apoptosis and Dcx-induced differentiation in vitro. In neurons, miR-134 reduces cell migration in vitro and in vivo in a Dcx-dependent manner. In differentiating neurons, miR-134 modulates process outgrowth in response to exogenous BMP-4 in a noggin-reversible manner. Taken together, we present Dcx and Chrdl-1 as new regulatory targets of miR-134 during embryonic, mouse, cortical, and neuronal differentiation and show a novel and previously undiscovered role for miR-134 in the stage-specific modulation of cortical development.
Assuntos
Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Córtex Cerebral/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , MicroRNAs/fisiologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Animais , Ciclo Celular/genética , Diferenciação Celular/genética , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Marcação de Genes/métodos , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/fisiologia , Neurônios/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismoRESUMO
Transfer RNA-derived fragments (tRFs) are emerging small noncoding RNAs that, although commonly altered in cancer, have poorly defined roles in tumorigenesis1. Here we show that pseudouridylation (Ψ) of a stem cell-enriched tRF subtype2, mini tRFs containing a 5' terminal oligoguanine (mTOG), selectively inhibits aberrant protein synthesis programmes, thereby promoting engraftment and differentiation of haematopoietic stem and progenitor cells (HSPCs) in patients with myelodysplastic syndrome (MDS). Building on evidence that mTOG-Ψ targets polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed isotope exchange proteomics to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains of PABPC1. Mechanistically, this hinders the recruitment of translational co-activator PABPC1-interacting protein 1 (PAIP1)3 and strongly represses the translation of transcripts sharing pyrimidine-enriched sequences (PES) at the 5' untranslated region (UTR), including 5' terminal oligopyrimidine tracts (TOP) that encode protein machinery components and are frequently altered in cancer4. Significantly, mTOG dysregulation leads to aberrantly increased translation of 5' PES messenger RNA (mRNA) in malignant MDS-HSPCs and is clinically associated with leukaemic transformation and reduced patient survival. These findings define a critical role for tRFs and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukaemia (AML).
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Fatores de Iniciação de Peptídeos/metabolismo , Pseudouridina , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
MicroRNAs (miRNAs) are small, noncoding RNA molecules with the ability to posttranscriptionally regulate gene expression via targeting the 3' untranslated region of messenger RNAs. miRNAs are critical for normal cellular functions such as the regulation of the cell cycle, differentiation, and apoptosis, and they target genes during embryonal and postnatal development, whereas their expression is unbalanced in various pathological states. Importantly, miRNAs are abundantly present in body fluids (e.g., blood), which are routinely examined in patients. These molecules circulate in free and exosome encapsulated forms, and can be efficiently detected and amplified by means of molecular biology tools such as real-time PCR. Together with relative stability, specificity, and reproducibility, they are seen as good candidates for early recognition of the onset of disease. Thus, miRNAs might be considered as biomarkers for many pathological states.
Assuntos
Biomarcadores/análise , Diabetes Mellitus/diagnóstico , Cardiopatias/diagnóstico , MicroRNAs/genética , Neoplasias/diagnóstico , Doenças Neurodegenerativas/diagnóstico , Diabetes Mellitus/genética , Cardiopatias/genética , Humanos , Neoplasias/genética , Doenças Neurodegenerativas/genéticaRESUMO
Small Cajal body-specific RNAs (scaRNAs) guide post-transcriptional modification of spliceosomal RNA and, while commonly altered in cancer, have poorly defined roles in tumorigenesis. Here, we uncover that SCARNA15 directs alternative splicing (AS) and stress adaptation in cancer cells. Specifically, we find that SCARNA15 guides critical pseudouridylation (Ψ) of U2 spliceosomal RNA to fine-tune AS of distinct transcripts enriched for chromatin and transcriptional regulators in malignant cells. This critically impacts the expression and function of the key tumor suppressors ATRX and p53. Significantly, SCARNA15 loss impairs p53-mediated redox homeostasis and hampers cancer cell survival, motility and anchorage-independent growth. In sum, these findings highlight an unanticipated role for SCARNA15 and Ψ in directing cancer-associated splicing programs.
RESUMO
Divalent copper and iron cations have been acknowledged for their catalytic roles in physiological processes critical for homeostasis maintenance. Being redox-active, these metals act as cofactors in the enzymatic reactions of electron transfer. However, under pathophysiological conditions, owing to their high redox potentials, they may exacerbate stress-induced injury. This could be particularly hazardous to the liver - the main body reservoir of these two metals. Surprisingly, the involvement of Cu and Fe in liver pathology still remains poorly understood. Hypoxic stress in the tissue may act as a stimulus that mobilizes these ions from their hepatic stores, aggravating the systemic injury. Since ischemia poses a serious complication in liver surgery (e.g. transplantation) we aimed to reveal the status of Cu and Fe via spectroscopic analysis of mouse ischemic liver tissue. Herein, we establish a novel non-surgical model of focal liver ischemia, achieved by applying light locally when a photosensitizer is administered systemically. Photodynamic treatment results in clear-cut areas of the ischemic hepatic tissue, as confirmed by ultrasound scans, mean velocity measurements, 3D modelling of vasculature and (immuno)histological analysis. For reference, we assessed the samples collected from the animals which developed transient systemic endotoxemic stress induced by a non-lethal dose of lipopolysaccharide. The electron paramagnetic resonance (EPR) spectra recorded in situ in the liver samples reveal a dramatic increase in the level of Cu adducts solely in the ischemic tissues. In contrast, other typical free radical components of the liver EPR spectra, such as reduced Riske clusters are not detected; these differences are not followed by changes in the blood EPR spectra. Taken together, our results suggest that local ischemic stress affects paramagnetic species containing redox-active metals. Moreover, because in our model hepatic vascular flow is impaired, these effects are only local (confined to the liver) and are not propagated systemically.