RESUMO
BCL-x is a master regulator of apoptosis whose pre-mRNA is alternatively spliced into either a long (canonical) anti-apoptotic Bcl-xL isoform, or a short (alternative) pro-apoptotic Bcl-xS isoform. The balance between these two antagonistic isoforms is tightly regulated and overexpression of Bcl-xL has been linked to resistance to chemotherapy in several cancers, whereas overexpression of Bcl-xS is associated to some forms of diabetes and cardiac disorders. The splicing factor RBM25 controls alternative splicing of BCL-x: its overexpression favours the production of Bcl-xS, whereas its downregulation has the opposite effect. Here we show that RBM25 directly and specifically binds to GQ-2, an RNA G-quadruplex (rG4) of BCL-x pre-mRNA that forms at the vicinity of the alternative 5' splice site leading to the alternative Bcl-xS isoform. This RBM25/rG4 interaction is crucial for the production of Bcl-xS and depends on the RE (arginine-glutamate-rich) motif of RBM25, thus defining a new type of rG4-interacting domain. PhenDC3, a benchmark G4 ligand, enhances the binding of RBM25 to the GQ-2 rG4 of BCL-x pre-mRNA, thereby promoting the alternative pro-apoptotic Bcl-xS isoform and triggering apoptosis. Furthermore, the screening of a combinatorial library of 90 putative G4 ligands led to the identification of two original compounds, PhenDH8 and PhenDH9, superior to PhenDC3 in promoting the Bcl-xS isoform and apoptosis. Thus, favouring the interaction between RBM25 and the GQ-2 rG4 of BCL-x pre-mRNA represents a relevant intervention point to re-sensitize cancer cells to chemotherapy.
Assuntos
Processamento Alternativo , Precursores de RNA , Apoptose , Isoformas de Proteínas/genética , Precursores de RNA/genética , Sítios de Splice de RNA , HumanosRESUMO
Splicing is functionally coupled to transcription, linking the rate of RNA polymerase II (Pol II) elongation and the ability of splicing factors to recognize splice sites (ss) of various strengths. In most cases, slow Pol II elongation allows weak splice sites to be recognized, leading to higher inclusion of alternative exons. Using CFTR alternative exon 9 (E9) as a model, we show here that slowing down elongation can also cause exon skipping by promoting the recruitment of the negative factor ETR-3 onto the UG-repeat at E9 3' splice site, which displaces the constitutive splicing factor U2AF65 from the overlapping polypyrimidine tract. Weakening of E9 5' ss increases ETR-3 binding at the 3' ss and subsequent E9 skipping, whereas strengthening of the 5' ss usage has the opposite effect. This indicates that a delay in the cotranscriptional emergence of the 5' ss promotes ETR-3 recruitment and subsequent inhibition of E9 inclusion.
Assuntos
Processamento Alternativo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Éxons , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Sítios de Splice de RNA/fisiologia , Ribonucleoproteínas/metabolismo , Sítios de Ligação , Proteínas CELF , Células CACO-2 , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células HEK293 , Humanos , Modelos Genéticos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Fator de Processamento U2AF , Transcrição GênicaRESUMO
CBS encodes a pyridoxal 5'-phosphate-dependent enzyme that catalyses the condensation of homocysteine and serine to form cystathionine. Due to its implication in some cancers and in the cognitive pathophysiology of Down syndrome, the identification of pharmacological inhibitors of this enzyme is urgently required. However, thus far, attempts to identify such molecules have only led to the identification of compounds with low potency and limited selectivity. We consequently developed an original, yeast-based screening method that identified three FDA-approved drugs of the 8-hydroxyquinoline family: clioquinol, chloroxine and nitroxoline. These molecules reduce CBS enzymatic activity in different cellular models, proving that the molecular mechanisms involved in yeast phenotypic rescue are conserved in mammalian cells. A combination of genetic and chemical biology approaches also revealed the importance of copper and zinc intracellular levels in the regulation of CBS enzymatic activity-copper promoting CBS activity and zinc inhibiting its activity. Taken together, these results indicate that our effective screening approach identified three new potent CBS inhibitors and provides new findings for the regulation of CBS activity, which is crucial to develop new therapies for CBS-related human disorders.
Assuntos
Cistationina beta-Sintase , Saccharomyces cerevisiae , Animais , Cobre , Cistationina beta-Sintase/genética , Humanos , Mamíferos , Oxiquinolina/farmacologia , Fosfato de Piridoxal , ZincoRESUMO
We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.
Assuntos
Neoplasias do Colo/genética , Ribonucleoproteína Nuclear Heterogênea A1/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Processamento Alternativo , Sítios de Ligação/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/metabolismo , Éxons , Células HCT116 , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Íntrons , Ligação Proteica/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
BACKGROUND: Colon cancer occurrence is increasing worldwide, making it the third most frequent cancer. Although many therapeutic options are available and quite efficient at the early stages, survival is strongly decreased when the disease has spread to other organs. The identification of molecular markers of colon cancer is likely to help understanding its course and, eventually, to uncover novel genes to be targeted by drugs. In this study, we compared gene expression in a set of 95 human colon cancer samples to that in 19 normal colon mucosae, focusing on 401 genes from 5 selected pathways (Apoptosis, Cancer, Cholesterol metabolism and lipoprotein signaling, Drug metabolism, Wnt/beta-catenin). Deregulation of mRNA levels largely matched that of proteins, leading us to build in silico protein networks, starting from mRNA levels, to identify key proteins central to network activity. RESULTS: Among the analyzed genes, 10.5% (42) had no reported link with colon cancer, including the SFRP1, IGF1 and ADH1B (down), and MYC and IL8 (up), whose encoded proteins were most interacting with other proteins from the same or even distinct networks. Analyzing all pathways globally led us to uncover novel functional links between a priori unrelated or rather remotely connected pathways, such as the Drug metabolism and the Cancer pathways or, even more strikingly, between the Cholesterol metabolism and lipoprotein signaling and the Cancer pathways. In addition, we analyzed the responsiveness of some of the deregulated genes essential to network activities, to chemotherapeutic agents used alone or in presence of Lovastatin, a lipid-lowering drug. Some of these treatments could oppose the deregulations occurring in cancer samples, including those of the CHECK2, CYP51A1, HMGCS1, ITGA2, NME1 or VEGFA genes. CONCLUSIONS: Our network-based approach allowed discovering genes not previously known to play regulatory roles in colon cancer. Our results also showed that selected drug treatments might revert the cancer-specific deregulation of genes playing prominent roles within the networks operating to maintain colon homeostasis. Among those genes, some could constitute novel testable targets to eliminate colon cancer cells, either directly or, potentially, through the use of lipid-lowering drugs such as statins, in association with selected anticancer drugs.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Perfilação da Expressão Gênica , Terapia de Alvo Molecular , Mapas de Interação de Proteínas/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Células HT29 , Humanos , Lovastatina/farmacologia , Lovastatina/uso terapêuticoRESUMO
Deregulation of the oxidative cascade of poly-unsaturated fatty acids (PUFAs) has been associated with several cancers, including chronic lymphocytic leukemia (B-CLL). Leukotriene B4 (LTB4), a metabolite of arachidonic acid (AA), is produced by B-CLL and contributes to their survival. The aim of the present study was to analyze the activity of the oxidative cascade of PUFAs in B-CLL. Purified B cells from patients and normal B CD5 positive cells were subjected to flow cytometry, Western-blot and RT-qPCR analyses. LTB4 plasma and intracellular concentrations were determined by ELISA. Our results showed that aggressive B-CLL tumor cells, i.e. cells with an annual proliferation index above 2, over-expressed calcium-dependent and calcium-independent phospholipases A2 (cPLA2-alpha and iPLA2-beta, respectively), 5-lipoxygenase (5LOX) and leukotriene A4 hydroxylase (LTA4H). Intracellular LTB4 levels were lower in the most aggressive cells than in cells with a smaller proliferation index, despite equivalent plasma levels, and lower expression of cytochrome P450 4F3A (CYP4F3A), one major enzyme involved in LTB4 inactivation. Since BLT2, a LTB4 membrane receptor was also more often expressed on aggressive tumor cells, and since a BLT2 inhibitor significantly impaired B-CLL viability in vitro, we propose that LTB4 was efficiently trapped onto BLT2 present on aggressive tumors, thereby eliciting an autocrine response. Taken together our results demonstrate a major deregulation of the pathway leading to LTB4 synthesis and degradation in B-CLL cells, and provide a framework for understanding how these modifications promote cell survival and proliferation, especially in the most aggressive BCLL.
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Alternative pre-mRNA splicing (AS) widely expands proteome diversity through the combinatorial assembly of exons. The analysis of AS on a large scale, by using splice-sensitive microarrays, is a highly efficient method to detect the majority of known and predicted alternative transcripts for a given gene. The response to targeted anticancer therapies cannot easily be anticipated without prior knowledge of the expression, by the tumor, of target proteins or genes. To analyze, in depth, transcript structure and levels for genes involved in these responses, including AKT1-3, HER1-4, HIF1A, PIK3CA, PIK3R1-2, VEGFA-D and PIR, we engineered a dedicated gene chip with coverage of an average 185 probes per gene and, especially, exon-exon junction probes. As a proof of concept, we demonstrated the ability of such a chip to detect the effects of over-expressed SRSF2 RNA binding protein on the structure and abundance of mRNA products in H358 lung cancer cells conditionally over-expressing SRSF2. Major splicing changes were observed, including in HER1/EGFR pre-mRNA, which were also seen in human lung cancer samples over-expressing the SRSF2 protein. In addition, we showed that variations in HER1/EGFR pre-mRNA splicing triggered by SRSF2 overexpression in H358 cells resulted in a drop in HER1/EGFR protein level, which correlated with increased sensitivity to gefitinib, an EGFR tyrosine kinase inhibitor. We propose, therefore, that this novel tool could be especially relevant for clinical applications, with the aim to predict the response before treatment.
Assuntos
Terapia de Alvo Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Precursores de RNA/genética , Adenocarcinoma/genética , Processamento Alternativo , Western Blotting , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase em Tempo Real , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-ArgininaRESUMO
BACKGROUND: Microsatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels. RESULTS: Our results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer. CONCLUSIONS: Overall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.
Assuntos
Processamento Alternativo , Neoplasias Colorretais , Instabilidade de Microssatélites , Fator de Processamento U2AF , Neoplasias Colorretais/genética , Humanos , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Mutação , Sítios de Ligação , ÉxonsRESUMO
Cancers that belong to the microsatellite instability (MSI) class can account for up to 15% of all cancers of the digestive tract. These cancers are characterized by inactivation, through the mutation or epigenetic silencing of one or several genes from the DNA MisMatch Repair (MMR) machinery, including MLH1, MLH3, MSH2, MSH3, MSH6, PMS1, PMS2 and Exo1. The unrepaired DNA replication errors turn into mutations at several thousand sites that contain repetitive sequences, mainly mono- or dinucleotides, and some of them are related to Lynch syndrome, a predisposition condition linked to a germline mutation in one of these genes. In addition, some mutations shortening the microsatellite (MS) stretch could occur in the 3'-intronic regions, i.e., in the ATM (ATM serine/threonine kinase), MRE11 (MRE11 homolog) or the HSP110 (Heat shock protein family H) genes. In these three cases, aberrant pre-mRNA splicing was observed, and it was characterized by the occurrence of selective exon skipping in mature mRNAs. Because both the ATM and MRE11 genes, which as act as players in the MNR (MRE11/NBS1 (Nibrin)/RAD50 (RAD50 double strand break repair protein) DNA damage repair system, participate in double strand breaks (DSB) repair, their frequent splicing alterations in MSI cancers lead to impaired activity. This reveals the existence of a functional link between the MMR/DSB repair systems and the pre-mRNA splicing machinery, the diverted function of which is the consequence of mutations in the MS sequences.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Instabilidade de Microssatélites , Humanos , Precursores de RNA , Mutação , Reparo do DNA , Neoplasias Colorretais Hereditárias sem Polipose/genéticaRESUMO
Resistance of carcinoma cells to anoikis, apoptosis that is normally induced by loss of cell-to-extracellular matrix adhesion, is thought to be essential for the ability of these cells to form primary tumors, invade adjacent tissues, and metastasize to distant organs. Current knowledge about the mechanisms by which cancer cells evade anoikis is far from complete. In an effort to understand these mechanisms, we found that ras, a major oncogene, down-regulates protease caspase-2 (which initiates certain steps of the cellular apoptotic program) in malignant human and rat intestinal epithelial cells. This down-regulation could be reversed by inhibition of a protein kinase Mek, a mediator of Ras signaling. We also found that enforced down-regulation of caspase-2 in nonmalignant intestinal epithelial cells by RNA interference protected them from anoikis. Furthermore, the reversal of the effect of Ras on caspase-2 achieved by the expression of exogenous caspase-2 in detached ras-transformed intestinal epithelial cells promoted well established apoptotic events, such as the release of the pro-apoptotic mitochondrial factors cytochrome c and HtrA2/Omi into the cytoplasm of these cells, significantly enhanced their anoikis susceptibility, and blocked their long term growth in the absence of adhesion to the extracellular matrix. Finally, the blockade of the effect of Ras on caspase-2 substantially suppressed growth of tumors formed by the ras-transformed cells in mice. We conclude that ras-induced down-regulation of caspase-2 represents a novel mechanism by which oncogenic Ras protects malignant intestinal epithelial cells from anoikis, promotes their anchorage-independent growth, and allows them to form tumors in vivo.
Assuntos
Caspase 2/biossíntese , Transformação Celular Neoplásica/metabolismo , Cisteína Endopeptidases/biossíntese , Regulação para Baixo , Células Epiteliais/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas ras/metabolismo , Animais , Anoikis/genética , Caspase 2/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cisteína Endopeptidases/genética , Citocromos c/genética , Citocromos c/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/patologia , Células Epiteliais/patologia , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Mucosa Intestinal/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/genética , Ratos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais/genética , Proteínas ras/genéticaRESUMO
Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF65 and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Linhagem Celular , Éxons , Humanos , Íntrons , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Sequências Repetitivas de Ácido Nucleico , Ribonucleoproteínas/metabolismo , Spliceossomos/metabolismo , Fator de Processamento U2AFRESUMO
Cytochrome P450 4F3 (CYP4F3), originally identified as one of the leukotriene B4 ω-hydroxylases, belongs to a CYP gene family that comprises several members, which participate in the metabolism of various endobiotics, as well as some xenobiotics. The CYP4F gene family is clustered in a 0.5-Mb stretch of genomic DNA on the p13 region of chromosome 19. Apart from the ω-hydroxylation of leukotriene B4 and prostaglandins, CYP4F3 is the main catalyst in the oxidation of fatty acid epoxides. CYP4F3 expression results from the synthesis of two distinct enzymes, CYP4F3A and CYP4F3B, which originate from the alternative splicing of a single pre-mRNA precursor molecule. Remarkably, the selection of either isoform is part of a tissue-specific control through which CYP3F3A is mostly expressed in leukocytes and CYP4F3B mostly in the liver. Recently, CYP4F3 single nucleotide polymorphisms have been incriminated in the onset of pathologies, including celiac or Crohn's diseases. Although much has been discovered in the regulation and function of CYP4F2, the closest CYP4F subfamily member, analyses of CYP4F3 enzymes lag somewhat behind in the field of our knowledge. In this short review, emphasis will be placed on the regulation and the functional roles of human CYP4F3.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Família 4 do Citocromo P450 , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Metabolismo dos Lipídeos/genética , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Xenobióticos/metabolismoRESUMO
Cell culture is an important and necessary technology in oncology research. Currently, two-dimensional (2D) cell culture models are the most widely used, but they cannot reproduce the complexity and pathophysiology of tumors in vivo. This may be a major cause of the high rate of attrition of anticancer drugs entering clinical trials, the rate of new anticancer drugs entering the market being less than 5 %. One way to improve the success of new cancer drugs in the clinic is based on the use of three-dimensional (3D) cell culture models, more able to represent the complex environment and architecture of tumors. These 3D culture systems are also a powerful research tool for modeling the evolution of cancer from early stages to metastasis. Spheroids and organoids, the most adaptable models among 3D culture systems, are beginning to be used in pharmaceutical research and personalized medicine. In this article, we review the use of spheroids and organoids by highlighting their differences, discussing their impact on drug development, and looking at future challenges.
Assuntos
Antineoplásicos/uso terapêutico , Técnicas de Cultura de Células em Três Dimensões/métodos , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Organoides , Esferoides Celulares , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Modelos Biológicos , Neoplasias/patologia , Organoides/efeitos dos fármacos , Organoides/patologia , Medicina de Precisão , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Gastric cancer (GC) is the third cause of cancer-related mortality worldwide and is often diagnosed at advanced stages of the disease. This makes the development of more comprehensive models and efficient treatments crucial. One option is based on repurposing already marketed drugs as adjuvants to chemotherapy. Accordingly, we have previously developed the combination of docetaxel and the cholesterol-lowering drug, lovastatin, as a powerful trigger of HGT-1 human GC cells' apoptosis using 2D cultures. Because 3D models, known as spheroids, are getting recognized as possibly better suited than 2Ds in toxicological research, we aimed to investigate the efficacy of this drug combination with such a model. We established monocellular spheroids from two human (GC) cell lines, HGT-1 and AGS, and bicellular spheroids from these cells mixed with cancer-associated fibroblasts. With these, we surveyed drug-induced cytotoxicity with MTT assays. In addition, we used the Incucyte live imaging and analysis system to follow spheroid growth and apoptosis. Taken together, our results showed that the lovastatin + docetaxel combination was an efficient strategy to eliminate GC cells grown in 2D or 3D cultures, lending further support in favor of repurposing lovastatin as an adjuvant to taxane-based anticancer treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Microscopia Intravital/métodos , Esferoides Celulares/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Fibroblastos Associados a Câncer , Técnicas de Cultura de Células em Três Dimensões , Linhagem Celular Tumoral , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Reposicionamento de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Lovastatina/farmacologia , Lovastatina/uso terapêutico , Neoplasias Gástricas/patologiaRESUMO
Gastric cancer (GC) is highly deadly. Three-dimensional (3D) cancer cell cultures, known as spheroids, better mimic tumor microenvironment (TME) than standard 2D cultures. Cancer-associated fibroblasts (CAF), a major cellular component of TME, promote or restrain cancer cell proliferation, invasion and resistance to drugs. We established spheroids from two human GC cell lines mixed with human primary CAF. Spheroid organization, analyzed by two-photon microscopy, showed CAF in AGS/CAF spheroids clustered in the center, but dispersed throughout in HGT-1/CAF spheroids. Such differences may reflect clonal specificities of GC cell lines and point to the fact that GC should be considered as a highly personalized disease.
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BACKGROUND: Statins have long been used as anti-hypercholesterolemia drugs, but numerous lines of evidence suggest that they may also bear anti-tumour potential. We have recently demonstrated that it was possible to isolate cancer cells adapted to growth in the continuous presence of lovastatin. These cells grew more slowly than the statin-sensitive cells of origin. In the present study, we compared the ability of both statin-sensitive and statin-resistant cells to give rise to tumours in Nude mice. METHODS: HGT-1 human gastric cancer cells and L50 statin-resistant derivatives were injected subcutaneously into Nude mice and tumour growth was recorded. At the end of the experiment, tumours were recovered and marker proteins were analyzed by western blotting, RT-PCR and immunohistochemistry. RESULTS: L50 tumours grew more slowly, showed a strong decrease in cyclin B1, over-expressed collagen IV, and had reduced laminin 332, VEGF and CD34 levels, which, collectively, may have restricted cell division, cell adhesion and neoangiogenesis. CONCLUSIONS: Taken together, these results showed that statin-resistant cells developed into smaller tumours than statin-sensitive cells. This may be reflective of the cancer restricting activity of statins in humans, as suggested from several retrospective studies with subjects undergoing statin therapy for several years.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lovastatina/farmacologia , Neoplasias Gástricas/prevenção & controle , Animais , Western Blotting , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Colágeno/metabolismo , Ciclina B1/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismo , CalininaRESUMO
Alternative splicing (AS) of pre-messenger RNAs is a major process contributing to both transcriptome and proteome diversity in various physiological and pathological situations. There is also accumulating evidence that various stresses impact on AS. In particular, recent analyses of the transcriptome reveal large numbers of AS events that are regulated by genotoxic stress inducers like radiations and chemotherapeutic agents. Many AS events have the potential to affect the relative production of protein isoforms with different activities, as shown in the case of several genes involved in apoptosis. There is also increasing evidence that stresses induce "non-productive" splice variants, leading to a decrease in gene expression levels or preventing increases in protein levels despite transcriptional stimulation. This is typically achieved by the production of splice variants that are subject to nonsense-mediated decay. In addition, recent studies suggest that pre-mRNA splicing efficiency or fidelity may be altered by stresses. For example, various genotoxic agents induce multiple exon skipping in MDM2 transcripts, thereby preventing the production of the main p53-ubiquitin ligase and favoring p53 activity in response to genotoxic agents. In terms of mechanisms, stresses can impact on pre-mRNA splicing by inducing post-translational modifications and subcellular redistribution of splicing factors, or by targeting the communication between the splicing and transcription machineries. Altogether, these data suggest that splicing regulatory networks play a key role in the cellular responses triggered by stresses.
Assuntos
Processamento Alternativo , Dano ao DNA , Isoformas de Proteínas/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Variação Genética , Humanos , Mutagênicos , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-mdm2/genética , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Transcrição Gênica , Transcriptoma , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.
Assuntos
Antineoplásicos/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Peptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos/química , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.