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This study investigated the neurological effects of residual ground-water levels of thiacloprid on the non-target organism Caenorhabditis elegans. Nematodes treated with thiacloprid showed a dose-dependent and significantly increased twitch response at concentrations above 50 ng mL-1 that disabled their forward locomotion in liquid culture. In comparison with untreated controls, 10 ng mL-1 thiacloprid perturbed the chemosensory ability of C. elegans such that the nematodes no longer demonstrated positive chemotaxis towards a NaCl chemo-attractant, reducing their chemotaxis index from +0.48 to near to zero. Nematodes also exhibited a locomotion characteristic of those devoid of chemo-attraction, making significantly more pirouetting turns of ≥90° than the untreated controls. Compared to the untreated controls, expression of the endocytosis-associated gene, Rab-10, was also increased in C. elegans that had developed to adulthood in the presence of 10 ng mL-1 thiacloprid, suggesting their active engagement in increased recycling of affected cellular components, such as their nAChRs. Thus, even residual, low levels of this less potent neonicotinoid that may be found in field ground-water had measurable effects on a beneficial soil organism which may have environmental and ecological implications that are currently poorly understood.
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Caenorhabditis elegans/efeitos dos fármacos , Inseticidas/toxicidade , Neonicotinoides/toxicidade , Resíduos de Praguicidas/análise , Tiazinas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Água Subterrânea/química , Locomoção/efeitos dos fármacos , Testes de ToxicidadeRESUMO
The peer review process is a fundamental aspect of modern scientific paper publishing, underpinning essential quality control. First conceptualised in the 1700s, it is an iterative process that aims to elevate scientific literature to the highest standards whilst preventing publication of scientifically unsound, potentially misleading, and even plagiarised information. It is widely accepted that the peer review of scientific papers is an irreplaceable and fundamental aspect of the research process. However, the rapid growth of research and technology has led to a huge increase in the number of publications. This has led to increased pressure on the peer review system. There are several established peer review methodologies, ranging from single and double blind to open and transparent review, but their implementation across journals and research fields varies greatly. Some journals are testing entirely novel approaches (such as collaborative reviews), whilst others are piloting changes to established methods. Given the unprecedented growth in publication numbers, and the ensuing burden on journals, editors, and reviewers, it is imperative to improve the quality and efficiency of the peer review process. Herein we evaluate the peer review process, from its historical origins to current practice and future directions.
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Revisão da Pesquisa por Pares , Humanos , Pesquisa Biomédica/tendências , Pesquisa Biomédica/normas , História do Século XXI , Revisão da Pesquisa por Pares/tendências , Revisão da Pesquisa por Pares/normas , Publicações Periódicas como Assunto , Editoração/normas , Editoração/tendências , Controle de QualidadeRESUMO
RNA biology has revolutionized cancer understanding and treatment, especially in endocrine-related malignancies. This editorial highlights RNA's crucial role in cancer progression, emphasizing its influence on tumor heterogeneity and behavior. Processes like alternative splicing and noncoding RNA regulation shape cancer biology, with microRNAs, long noncoding RNAs, and circular RNAs orchestrating gene expression dynamics. Aberrant RNA signatures hold promise as diagnostic and prognostic biomarkers in endocrine-related cancers. Recent findings, such as aberrant PI3Kδ splice isoforms and epithelial-mesenchymal transition-related lncRNA signatures, unveil potential therapeutic targets for personalized treatments. Insights into m6A-associated lncRNA prognostic models and the function of lncRNA LINC00659 in gastric cancer represents ongoing research in this field. As understanding of RNA's role in cancer expands, personalized therapies offer transformative potential in managing endocrine-related malignancies. This signifies a significant stride towards precision oncology, fostering innovation for more effective cancer care.
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Neoplasias , Humanos , Neoplasias/genética , Neoplasias/terapia , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante/genética , Biomarcadores Tumorais/genética , MicroRNAs/genética , Medicina de Precisão/métodos , RNA/genética , RNA Circular/genética , AnimaisRESUMO
Following infection or exposure to therapeutic agents, an aggressive immune response may result, termed cytokine storm (CS) or cytokine release syndrome. Here the innate immune system becomes uncontrolled, leading to serious consequences including possible death. Patients surviving CS are at greater risk for de novo tumorigenesis, but it is unclear if any specific cytokines are directly responsible for this outcome. De novo tumorigenesis has been observed in donated cells exposed to CS following haematopoietic stem cell transplant (HSCT). Modelling HSCT, we firstly demonstrated the release of CS levels from the HS-5 human bone marrow stromal cell line, post-exposure to chemotherapy. We then exposed the TK6 lymphoblast cell line to healthy and storm doses of IL-6 and measured increased genotoxicity via the micronucleus assay. During HSCT, haematopoietic cells are exposed to a complex mix of cytokines, so to determine if IL-6 was integral in a chemotherapy-induced bystander effect, we attempted to inhibit IL-6 from HS-5 cells using resatorvid or siRNA, treated with chlorambucil or mitoxantrone, and then co-cultured with bystander TK6 cells. Whilst resatorvid did not reduce IL-6 and did not reduce micronuclei in the bystander TK6 cells, siRNA inhibition reduced IL-6 to healthy in vivo levels, and micronuclei aligned with untreated controls. Our data suggests that exposure to high IL-6 (in the absence of inflammatory cells) has potential to induce genetic damage and may contribute to de novo tumorigenesis post-CS. We suggest that for individuals with a pro-inflammatory profile, anti-IL-6 therapy may be an appropriate intervention to prevent complications post-CS.
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Formalin fixed and paraffin embedded (FFPE) human tissue collections are an invaluable resource for retrospective gene expression studies. However formalin fixation results in chemical modification of RNA and increased RNA degradation. This can affect RNA yield and quality. A critical step when analysing gene expression is the conversion of RNA to complementary DNA (cDNA) using a reverse transcriptase (RT) enzyme. FFPE derived RNA may affect the performance and efficiency of the RT enzyme and cDNA synthesis. We directly compared three commonly used FFPE RNA isolation methods and measured RNA yield, purity and integrity. We also assessed the effectiveness of three commercially available Moloney Murine Leukemia Virus (M-MLV) RTs on cDNA synthesis and gene expression sensitivity when using FFPE RNA as a template. Our results show that gene detection sensitivity is dependent on the isolation method, RT and length of the PCR amplicon (<200bp) when using FFPE RNA. The use of an M-MLV RT enzyme with reduced RNaseH activity gave significantly increased qRT-PCR sensitivity when using FFPE RNA derived from prostate tissue. The choice of RT can also affect perceived changes in target gene expression and thus the same RT should be used when attempting to reproduce results from different studies. This study highlights the need to optimise and evaluate RNA isolation methods and RTs when using FFPE RNA as a template in order to maximise a successful outcome in PCR applications.
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Perfilação da Expressão Gênica/métodos , Vírus da Leucemia Murina de Moloney/enzimologia , Inclusão em Parafina , Neoplasias da Próstata/genética , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Formaldeído , Humanos , Limite de Detecção , Masculino , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribonuclease H/metabolismo , Sensibilidade e EspecificidadeRESUMO
AS (alternative splicing) and its role in disease, especially cancer, has come to forefront in research over the last few years. Alterations in the ratio of splice variants have been widely observed in cancer. Splice variants of cancer-associated genes have functions that can alter cellular phenotype, ultimately altering metastatic potential. As metastases are the cause of approximately 90% of all human cancer deaths, it is crucial to understand how AS is dysregulated in metastatic disease. We highlight some recent studies into the relationship between altered AS of key genes and the initiation of prostate cancer metastasis.
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Neoplasias da Próstata/genética , Splicing de RNA/genética , Processamento Alternativo/genética , Animais , Progressão da Doença , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MasculinoRESUMO
SRPK1 (serine-arginine protein kinase 1) is a protein kinase that specifically phosphorylates proteins containing serine-arginine-rich domains. Its substrates include a family of SR proteins that are key regulators of mRNA AS (alternative splicing). VEGF (vascular endothelial growth factor), a principal angiogenesis factor contains an alternative 3' splice site in the terminal exon that defines a family of isoforms with a different amino acid sequence at the C-terminal end, resulting in anti-angiogenic activity in the context of VEGF165-driven neovascularization. It has been shown recently in our laboratories that SRPK1 regulates the choice of this splice site through phosphorylation of the splicing factor SRSF1 (serine/arginine-rich splicing factor 1). The present review summarizes progress that has been made to understand how SRPK1 inhibition may be used to manipulate the balance of pro- and anti-angiogenic VEGF isoforms in animal models in vivo and therefore control abnormal angiogenesis and other pathophysiological processes in multiple disease states.
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Proteínas Serina-Treonina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Processamento Alternativo/genética , Animais , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF(165), and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF(165)b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF(165) and less VEGF(165)b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.
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Processamento Alternativo , Inibidores da Angiogênese/biossíntese , RNA Mensageiro/biossíntese , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Inibidores da Angiogênese/genética , Animais , Linhagem Celular Transformada , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Neovascularização Retiniana/genética , Neovascularização Retiniana/terapia , Fatores de Processamento de Serina-Arginina , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.
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Quinases Ciclina-Dependentes/antagonistas & inibidores , Terapia de Alvo Molecular , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Processamento Alternativo/genética , Animais , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos Nus , Invasividade Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA-Seq , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The zinc finger transcription factor Wilms tumour protein (WT 1) is known for its essential involvement in the development of the genitourinary system as well as of other organs and tissues. WT 1 is capable of selectively binding either DNA or mRNA targets. A KTS insertion due to alternative splicing between the zinc fingers 3 and 4 and an unconventional zinc finger 1 are the unique features that distinguish WT 1 from classical DNA-binding C(2)H(2)-type zinc finger proteins. The DNA binding characteristics of WT 1 are well studied. Due to lack of information about its native RNA targets, no extensive research has been directed at how WT 1 binds RNA. Using surface plasmon resonance, this study attempts to understand the binding behaviour of WT 1 zinc fingers with its recently reported and first putative mRNA target, ACT 34, whose stem-loop structure is believed to be critical for the interactions with WT 1. We have analysed the interactions of five WT 1 zinc finger truncations with wild-type ACT 34 and four variants. Our results indicate that WT 1 zinc fingers bind ACT 34 in a specific manner, and that this occurs as interplay of all four zinc fingers. We also report that a sensitive kinetic balance, which is equilibrated by both zinc finger 1 and KTS, regulates the interaction with ACT 34. The stem-loop and the flanking nucleotides are important elements for specific recognition by WT 1 zinc fingers.
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Actinina/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Proteínas WT1/metabolismo , Cinética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , RNA/metabolismo , Ressonância de Plasmônio de Superfície , Fatores de Transcrição/genética , Proteínas WT1/genética , Dedos de Zinco/genéticaRESUMO
WT1 was first described in 1990 as a tumour suppressor gene associated with Wilms tumour (nephroblastoma). It encodes a typical transcription factor with four C(2)-H(2) zinc fingers in the C-terminus. However WT1 is surprisingly complex at multiple levels: it is involved in the development of several organ systems; and is both a tumour suppressor and oncogene. Here we review evidence that has accumulated over the past decade to suggest that as well as binding DNA, WT1 also binds mRNA targets via its zinc fingers and interacts with several splice factors. WT1's first reported post-transcriptional function is also reviewed. WT1's complex roles in development and disease now need to be understood in terms of both DNA and mRNA targets.
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Processamento Pós-Transcricional do RNA , Proteínas WT1/fisiologia , Processamento Alternativo , Animais , Regulação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , RNA Mensageiro/metabolismo , Ribonucleoproteínas/química , Dedos de Zinco/fisiologiaRESUMO
A hypoxic environment can be defined as a region of the body or the whole body that is deprived of oxygen. Hypoxia is a feature of many diseases, such as cardiovascular disease, tissue trauma, stroke, and solid cancers. A loss of oxygen supply usually results in cell death; however, when cells gradually become hypoxic, they may survive and continue to thrive as described for conditions that promote metastatic growth. The role of hypoxia in these pathogenic pathways is therefore of great interest, and understanding the effect of hypoxia in regulating these mechanisms is fundamentally important. This chapter gives an extensive overview of these mechanisms. Moreover, given the challenges posed by tumor hypoxia we describe the current methods to simulate and detect hypoxic conditions followed by a discussion on current and experimental therapies that target hypoxic cells.
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Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/fisiopatologia , Neoplasias/patologia , Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/terapia , Oxigênio/metabolismoRESUMO
The oncogene ERG encodes an ETS family transcription factor and is implicated in blood, vascular, and bone development and in prostate, blood, and bone cancer. The ERG gene is alternatively spliced; of particular interest is its cassette exon 7b which adds 24 amino acids, in frame, to the transcriptional activation domain. Higher exon 7b inclusion rates are associated with increased cell proliferation and advanced prostate cancer. The 24 amino acids encoded by exon 7b show evolutionary conservation from humans to echinoderms, highlighting their functional importance. Throughout evolution, these 24 amino acids are encoded by a distinct short exon. Splice-switching oligonucleotides based on morpholino chemistry were designed to induce skipping of ERG exon 7b in MG63 osteosarcoma and VCaP prostate cancer cells. Induction of exon 7b skipping reduced cell proliferation and invasion, increased apoptosis in vitro, and reduced xenograft growth in vivo. We also show that ERG's exon 7b is required for the induction of tissue nonspecific alkaline phosphatase. Together, these findings show that the evolutionarily conserved cassette exon 7b is central to ERG's oncogenic properties.
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The modification of proteins is a key way to alter their activity and function. Often thiols, cysteine residues, on proteins are attractive targets for such modification. Assuming that the thiol group is accessible then reactions may take place with a range of chemicals found in cells. These may include reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), reactive nitrogen species such as nitric oxide (NO), hydrogen sulfide (H2S), or glutathione. Such modifications often are instrumental to important cellular signaling processes, which ultimately result in modification of physiology of the organism. Therefore, there is a need to be able to identify such modifications. There are a variety of techniques to find proteins which may be altered in this way but here the focus is on two approaches: firstly, the use of fluorescent thiol derivatives and the subsequent use of mass spectrometry to identify the thiols involved; secondly the confirmation of such changes using biochemical assays and genetic mutants. The discussion will be based on the use of two model organisms: firstly the plant Arabidopsis thaliana (both as cell cultures and whole plants) and secondly the nematode worm Caenorhabditis elegans. However, these tools, as described, may be used in a much wider range of biological systems, including human and human tissue cultures.
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Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Sulfeto de Hidrogênio/farmacologia , Espécies Reativas de Nitrogênio/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Poluentes Atmosféricos/farmacologia , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/metabolismo , Processamento de Proteína Pós-Traducional , Compostos de Sulfidrila/químicaRESUMO
Alternative splicing, first discovered in the 1970s, has emerged as one of the key generators of proteomic diversity. Not surprisingly, alternative splicing is increasingly linked to the etiology of cancer. This is illustrated by vascular endothelial growth factor (VEGF), the dominant angiogenic factor. Recently, an antiangiogenic family of VEGF isoforms was discovered, and termed VEGF(xxx)b. VEGF(xxx)b isoforms arise from an alternative 3' splice site in exon 8, and differ by a mere six amino acids at the C-terminus. These alternative six amino acids radically change the functional properties of VEGF. VEGF(xxx)b isoform expression is regulated in human tissues and development, and disregulated in many pathological states including cancer. Understanding what regulates VEGF(xxx)b alternative splicing, and therefore the balance of pro- and antiangiogenic isoforms is of great importance and will be explored in detail over the next few years.
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Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/genética , Processamento Alternativo , Animais , HumanosRESUMO
The oncogene ETS-related gene (ERG) encodes a transcription factor with roles in the regulation of haematopoiesis, angiogenesis, vasculogenesis, inflammation, migration and invasion. The ERG oncogene is activated in >50% of prostate cancer cases, generally through a gene fusion with the androgen-responsive promoter of transmembrane protease serine 2. Phosphatase and tensin homologue (PTEN) is an important tumour suppressor gene that is often inactivated in cancer. ERG overexpression combined with PTEN inactivation or loss is often associated with aggressive prostate cancer. The present study aimed to determine whether or not ERG regulates PTEN transcription directly. ERG was demonstrated to bind to the PTEN promoter and repress its transcription. ERG overexpression reduced endogenous PTEN expression, whereas ERG knockdown increased PTEN expression. The ability of ERG to repress PTEN may contribute to a more cancer-permissive environment.
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The WT1 gene is a key player in acute myeloid leukaemia, in which it is frequently over-expressed. WT1 encodes a multifunctional zinc finger protein transcription factor, which also binds mRNA. Thus increasing evidence suggests that WT1 works both at the DNA and mRNA level, not only in the urogenital system but also in other contexts. Nuclear poly(A)(+) mRNP particles were isolated by oligo(dT) chromatography from the human acute myeloid leukemia cell lines HL60 and K562, and analysed by Western blotting and 2D minigels. MALDI-TOF demonstrated the presence of hnRNP proteins, splice factors, and unexpectedly vimentin in the mRNP fraction. WT1 was also shown to be present in nuclear mRNP particles suggesting that in leukaemia, and by extension in all cancers in which it is involved, WT1 works both at the DNA and mRNA target level.
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Núcleo Celular/metabolismo , DNA/metabolismo , Leucemia Mieloide/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas WT1/metabolismo , Doença Aguda , DNA/genética , Eletroforese em Gel Bidimensional , Células HL-60 , Humanos , Células K562 , RNA Mensageiro/genética , Ribonucleoproteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas WT1/análiseRESUMO
Men who develop prostate cancer (PCa) increasingly have one of the co-morbidities associated with a Western lifestyle that are characterized by hyperinsulinemia, hyperglycemia and increased expression of insulin-like growth factors-I (IGF-I) and IGF-II. Each have been associated with poor prognosis and more aggressive cancers that exhibit increased metabolism and increased glucose uptake. The insulin receptor (IR) has two splice isoforms IR-A and IR-B: IR-A has a higher affinity for IGF-II comparable to that for insulin, whereas the IR-B isoform predominantly just binds to insulin. In this study, we assessed alterations in the IR-A and IR-B isoform ratio and associated changes in cell proliferation and migration of PCa cell lines following exposure to altered concentrations of glucose and treatment with IGF-II and insulin. We observed that where IR-B predominated insulin had a greater effect on migration than IGF-II and IGF-II was more effective when IR-A was the main isoform. With regard to proliferation IGF-II was more effective than insulin regardless of which isoform was dominant. We assessed the abundance of the IR isoforms both in vivo and in vitro and observed that the majority of the tissue samples and cell lines expressed more IR-A than IR-B. Alterations in the isoforms in response to changes in their hormonal milieu could have a profound impact on how malignant cells behave and play a role in promoting carcinogenesis. A greater understanding of the mechanisms underlying changes in alternative splicing of the IR may provide additional targets for future cancer therapies.
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The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy.
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Processamento Alternativo , Bevacizumab , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neovascularização Patológica/metabolismo , Proteínas de Ligação a Poli(A)/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células HEK293 , Células HeLa , Xenoenxertos , Humanos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteínas de Ligação a Poli(A)/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Antígeno-1 Intracelular de Células T , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVES: The proto-oncogene ETS-related gene (ERG) is consistently overexpressed in prostate cancer. Alternatively spliced isoforms of ERG have variable biological activities; inclusion of exon 11 (72 base pairs [bp]) is associated with aggressiveness and progression of disease. Exon 10 (81 bp) has also been shown to be alternatively spliced. Within this study, we assess whether ERG protein, messenger RNA (mRNA), and ERG splice isoform mRNA expression is altered as prostate cancer progresses. METHODS: Detection of the TMPRSS2-ERG fusion was done using direct methods (reverse transcription polymerase chain reaction [PCR] and fluorescence in situ hybridization) and indirect methods for ERG mRNA and protein expression using quantitative PCR and immunohistochemistry, respectively. A linear equation method was used to quantitatively determine relative proportions of ERG variants (ERG72/Δ72, ERG81/Δ81) for each sample. RESULTS: ERG mRNA and protein expression is increased in patients with advanced prostate cancer, with higher levels of ERG expression significantly associated with seminal vesicle invasion (stage pT3b) and biochemical recurrence. Genes involved in cell migration and invasiveness (matrix metalloproteinase 7, osteopontin, and septin 9) are increased in prostate cancers that overexpress ERG. In addition, there is a clear indication of increased retention of exons 10 and 11 in prostate cancer. CONCLUSIONS: Analysis of ERG and its variants may be valuable in determining prognosis and development of prostate cancer.