RESUMEN
In this study, two novel alternative splice variants of HER2, named HER2-PI9 and HER2-I12, were identified in breast cancer cell lines and breast tumour tissues. Whilst HER2-P19 arises from the inclusion of an 117 bp cassette-exon of intron 9 of HER2, HER2-I12 results from intron 12 inclusion. In silico analyses were performed to predict the amino acid sequences of these two HER2 novel variants. To confirm their protein expression, plasmid vectors were generated and transfected into the HER2 negative breast cancer cell line, MCF-7. Additionally, their functional properties in oncogenic signalling were confirmed. Expression of HER2-PI9 and HER2-I12 was successful and matched the in silico predictions. Importantly, these splice variants can modulate the phosphorylation levels of extracellular signal-related kinase 1/2 (ERK1/2) and Akt/protein kinase B (Akt) signalling in MCF-7 breast cancer cells. Enhanced cellular proliferation, migration and invasion were observed in the case of the HER2-I12 expressing model. In human tissues and breast carcinoma tumours both variants were present. This study reveals two novel splice variants of HER2. Additionally, the potential biological activity for HER2-PI9 and HER2-I12 in breast cancer cells is also reported..
Asunto(s)
Empalme Alternativo , Biomarcadores de Tumor/genética , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Receptor ErbB-2/genética , Apoptosis , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Proliferación Celular , Femenino , Humanos , Células Tumorales CultivadasRESUMEN
Ceramic orthopaedic implants are increasingly popular due to the need for robust total joint replacement implants that have a high success rate long-term and do not induce biological responses in patients. This study was designed to investigate the biological effects of ceramic nanopowders containing aluminium oxide or zirconium oxide to activate the human macrophage THP-1 cell line. In vitro investigation of pro-inflammatory gene expression and chemokine secretion was performed studied using RT-qPCR and ELISA, respectively. TLR4 inhibition, using a small-molecule inhibitor, was used to determine whether ceramic-mediated inflammation occurs in a similar manner to that of metals such as cobalt. THP-1 macrophages were primed with ceramics or LPS and then treated with ATP or ceramics, respectively, to determine whether these nanopowders are involved in the priming or activation of the NLRP3 inflammasome through IL-1ß secretion. Cells treated with ceramics significantly increased pro-inflammatory gene expression and protein secretion which was attenuated through TLR4 blockade. Addition of ATP to cells following ceramic treatment significantly increased IL-1ß secretion. Therefore, we identify the ability of ceramic metal oxides to cause a pro-inflammatory phenotype in THP-1 macrophages and propose the mechanism by which this occurs is primarily via the TLR4 pathway which contributes to inflammasome signalling.
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Óxido de Aluminio/farmacología , Cerámica , Inflamación/inducido químicamente , Nanopartículas/química , Polvos/farmacología , Circonio/farmacología , Artroplastia de Reemplazo de Cadera , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Fagocitosis , Células THP-1 , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismoRESUMEN
Overexpression of the HER2 receptor occurs in approximately 20% of breast cancer patients. HER2 positivity is associated with poor prognosis and aggressive tumour phenotypes, which led to rapid progress in HER2 targeted therapeutics and diagnostic testing. Whilst these advances have greatly increased patients' chances of survival, resistance to HER2 targeted therapies, be that intrinsic or acquired, remains a problem. Different forms of the HER2 protein exist within tumours in tandem and can display altered biological activities. Interest in HER2 variants in breast cancer increased when links between resistance to anti-HER2 therapies and a particular variant, Δ16-HER2, were identified. Moreover, the P100 variant potentially reduces the efficacy of the anti-HER2 therapy trastuzumab. Another variant, Herstatin, exhibits 'auto-inhibitory' behaviour. More recently, new HER2 variants have been identified and are currently being assessed for their pro- and anti-cancer properties. It is important when directing the care of patients to consider HER2 variants collectively. This review considers HER2 variants in the context of the tumour environment where multiple variants are co-expressed at altered ratios. This study also provides an up to date account of the landscape of HER2 variants and links this to patterns of resistance against HER2 therapies and treatment plans.
RESUMEN
The female climacteric or menopausal process characterised by reduced estrogen, associates with an increased risk of recurrent urinary tract infections (rUTIs) linked to uropathogenic Escherichia coli (UPEC). Clinically, topical vaginal estrogen treatment has a prophylactic effect against such infections. The aim of this study was to investigate, in vitro, the effects of a topical estrogen treatment on vaginal epithelial responses following challenge with E.coli flagellin mimicking an UPEC challenge. Immortalised vaginal epithelial cells (VK2 E6/E7), modelling the vaginal epithelium were treated with either 4 nM 17ß-estradiol (E) for seven days, 50 ng/ml E.coli flagellin (F) for 12 h, or 4 nM 17ß-estradiol plus 50 ng/ml flagellin (E + F(12 h)). RNA was analysed by microarray gene profiling using the Illumina HumanHT-12 v 4 Expression Beadchip. Following E + F treatments expression of genes encoding host defence molecules including DEFß4A, DEFB103A, LCN2 as well as those associated with keratinisation eg CNFN and SPRR family genes were significantly enhanced (P < 0.05) compared to either E or F treatments alone. Mutation of estrogen responsive elements (EREs) identified in the DEFß4 gene promoter abolished the augmented gene expression suggesting estrogen functioned directly through a regulatory mechanism involving ESR1/2. Ingenuity pathway analyses also suggested the pro-inflammatory cytokine IL-17A to regulate the vaginal host defences during infection. Pre-treating VK2 E6/E7 cells with estrogen (4 nM) and challenging with 1L-17A & F (12 h) significantly enhanced DEFß4, DEF103A and S100A7 expression (P < 0.05). Origins of vaginal IL-17 in vivo remain unclear, but patient biopsies support γδ T cells located within the vaginal epithelium. These data suggest that the vaginal antimicrobial response induced by flagellin activation of Toll-like Receptor 5 cell signalling is augmented following topical estrogen application.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Estrógenos/administración & dosificación , Flagelina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Vagina/fisiología , Administración Tópica , Proteínas de Escherichia coli/genética , Femenino , Flagelina/genética , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Vagina/efectos de los fármacos , Vagina/metabolismoRESUMEN
Myeloid cell leukemia-1 (Mcl -1) is one of the most frequently amplified genes in cancer, and its overexpression is associated with poor prognosis and drug resistance. As a member of the Bcl-2 family it is involved in the control of the mitochondrial (intrinsic) cell death pathway. Alternative splicing of the (Mcl-1) gene results in the expression of two functionally distinct proteins, the anti-apoptotic Mcl-1L (exon 2 included) and the pro-apoptotic Mcl-1S (exon 2 skipped). Our data shows that transfecting siRNAs that target hnRNP K and the hnRNP F/H family result in a switch in splicing towards the pro-apoptotic Mcl-1S. Specific binding sites for these and other Mcl-1 splicing factors were investigated and identified by RNA immunoprecipitation and through construction of a Mcl-1 minigene construct. Moreover, this study shows up to a 30 fold change in the levels of Mcl-1S can be achieved through double and triple knockdowns of the most significant RNA binding proteins involved in Mcl-1 splicing, as well as activation of the mitochondrial cell death pathway. Targeting the splicing process of Mcl-1 along with other apoptotic regulators provides an exciting new therapeutic target in cancer cells, and may provide a way to overcome therapy resistance.
Asunto(s)
Empalme Alternativo , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Apoptosis/genética , Sitios de Unión , Femenino , Técnicas de Inactivación de Genes , Humanos , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
The identification of the host defence peptides as target effectors in the innate defence of the uro-genital tract creates new translational possibilities for immunomodulatory therapies, specifically vaginal therapies to treat women suffering from rUTI, particularly those carrying the TLR5_C1174T SNP. Urinary tract infections (UTIs) are a microbial disease reported worldwide. Women are particularly susceptible with many suffering debilitating recurrent (r) infections. Treatment is by antibiotics, but such therapy is linked to antibiotic resistance and re-infection. This study explored the innate protective mechanisms of the urogenital tract with the aim of boosting such defences therapeutically. Modelling UTIs in vitro, human vaginal and bladder epithelial cells were challenged with uropathogenic Escherichia coli (CFT073) and microbial PAMPs including flagellin, LPS and peptidoglycan. Flagellin functioning via the TLR5/NFκB pathway was identified as the key UPEC virulence factor causing a significant increase (P < 0.05) in the production of the host-defence peptide (HDP), BD2. BD2-depleted urine samples from bladder infected mice supported increased UPEC growth, strengthening the significance of the HDPs in protecting the urogenital tissues from infection. Clinically, vaginal-douche BD2 concentrations were reduced (p < 0.05) in women suffering rUTIs, compared to age-matched healthy controls with concentrations further decreased (p < 0.05) in a TLR5392Stop SNP rUTI subgroup. Topical vaginal estrogen treatment increased (p < 0.001) BD2 concentrations in all women, including those carrying the SNP. These data identify therapeutic and antibiotic sparing roles for vaginal immunomodulatory agents that specifically target HDP induction, facilitate bacterial killing and disrupt the UPEC infection cycle.
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Infecciones por Escherichia coli/inmunología , Inmunidad Innata , Receptor Toll-Like 5/metabolismo , Infecciones Urinarias/inmunología , Vagina/inmunología , Vagina/microbiología , beta-Defensinas/metabolismo , Adulto , Anciano , Animales , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Femenino , Humanos , Ratones , Persona de Mediana Edad , Modelos Biológicos , Recurrencia , Escherichia coli Uropatógena/crecimiento & desarrollo , Escherichia coli Uropatógena/inmunología , Adulto JovenRESUMEN
The human epidermal growth factor receptor 2 (HER2)/receptor tyrosine-protein kinasebB-2 (ERBB2) is overexpressed in 20-30% of breast tumors leading to faster growing and more aggressive tumors. Alternative splicing generates a functionally distinct HER2 variant called Herstatin, which is produced by the inclusion of intron 8. Herstatin acts as a tumor suppressor by effectively blocking HER2 activity and cell proliferation, while promoting apoptosis. In the present study we investigated HER2 pre-mRNA regulatory sequences and splicing factors which regulate the alternative splicing of Herstatin. A Herstatin minigene, comprising exon 8/intron 8/exon 9 of HER2 was generated and subsequent in vitro splicing assays revealed that RNA secondary structure and somatic mutations did not impact on inclusion of intron 8. However, using RNase-assisted RNA chromatography, followed by mass spectrometry, we identified six RNA-binding proteins (splicing factors) that bind to RNA sequences surrounding exon 8/intron 8 and intron 8/exon 9 boundaries; these included hnRNP I, H1, D, A2/B1 and hnRNPA1 plus the SR protein SRSF1. Specifically, overexpression of hnRNP A1 significantly increased retention of intron 8 resulting in higher levels of Herstatin in SKBR3 breast cancer cells whereas SRSF1 only had a marginal effect in decreasing Herstatin but increased exogenous HER2 levels under these experimental conditions. In conclusion, we have identified the first splicing factors and regulatory sequences that are involved in the production of Herstatin.
Asunto(s)
Empalme Alternativo , Neoplasias de la Mama/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factores de Empalme de ARN/metabolismo , Receptor ErbB-2/genética , Proteínas Supresoras de Tumor/metabolismo , Apoptosis , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Exones , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Intrones , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein-RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Empalme Alternativo , Animales , Sitios de Unión , Exones , Intrones , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Dominios Proteicos , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Especificidad por SustratoRESUMEN
Cobalt-containing metal-on-metal hip replacements are associated with adverse reactions to metal debris (ARMD), including inflammatory pseudotumours, osteolysis, and aseptic implant loosening. The exact cellular and molecular mechanisms leading to these responses are unknown. Cobaltions (Co2+) activate human Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to Gram negative bacterial lipopolysaccharide (LPS).We investigated the effect of Co2+-mediated TLR4 activation on human microvascular endothelial cells (HMEC-1), focusing on the secretion of key inflammatory cytokines and expression of adhesion molecules. We also studied the role of TLR4 in Co2+-mediated adhesion molecule expression in MonoMac 6 macrophages.We show that Co2+ increases secretion of inflammatory cytokines, including IL-6 and IL-8, in HMEC-1. The effects are TLR4-dependent as they can be prevented with a small molecule TLR4 antagonist. Increased TLR4-dependent expression of intercellular adhesion molecule 1 (ICAM1) was also observed in endothelial cells and macrophages. Furthermore, we demonstrate for the first time that Co2+ activation of TLR4 upregulates secretion of a soluble adhesion molecule, sICAM-1, in both endothelial cells and macrophages. Although sICAM-1 can be generated through activity of matrix metalloproteinase-9 (MMP-9), we did not find any changes in MMP9 expression following Co2+ stimulation.In summary we show that Co2+ can induce endothelial inflammation via activation of TLR4. We also identify a role for TLR4 in Co2+-mediated changes in adhesion molecule expression. Finally, sICAM-1 is a novel target for further investigation in ARMD studies.
Asunto(s)
Cobalto/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Receptor Toll-Like 4/agonistas , Citocinas/metabolismo , Activación Enzimática , Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismoRESUMEN
Cobalt-chrome alloy is a widely used biomaterial in joint replacements, dental implants and spinal rods. Although it is an effective and biocompatible material, adverse reactions to metal debris (ARMD) have arisen in a minority of patients, particularly in those with metal-on-metal bearing hip replacements. There is currently no treatment for ARMD and once progressive, early revision surgery of the implant is necessary. Therapeutic agents to prevent, halt or reverse ARMD would therefore be advantageous. Cobalt ions activate Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to bacterial lipopolysaccharide (LPS) resulting in the production of pro-inflammatory cytokines and chemokines. We hypothesised that anti-TLR4 neutralising antibodies, reported to inhibit TLR4-mediated inflammation, could prevent the inflammatory response to cobalt ions in an in vitro macrophage cell culture model. This study shows that a monoclonal anti-TLR4 antibody inhibited cobalt-mediated increases in pro-inflammatory IL8, CCL20 and IL1A expression, as well as IL-8 secretion. In contrast, a polyclonal antibody did not prevent the effect of cobalt ions on either IL-8 or IL1A expression, although it did have a small effect on the CCL20 response. Interestingly, both antibodies inhibited cobalt-mediated neutrophil migration although the greater effect was observed with the monoclonal antibody. In summary our data shows that a monoclonal anti-TLR4 antibody can inhibit cobalt-mediated inflammatory responses while a polyclonal antibody only inhibits the effect of specific cytokines. Anti-TLR4 antibodies have therapeutic potential in ARMD although careful antibody design is required to ensure that the LPS response is preserved.
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Anticuerpos Monoclonales/farmacología , Cobalto/efectos adversos , Inflamación/prevención & control , Leucemia Monocítica Aguda/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidores , Anticuerpos Neutralizantes/farmacología , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Inflamación/inducido químicamente , Inflamación/patología , Mediadores de Inflamación/metabolismo , Leucemia Monocítica Aguda/patología , Macrófagos/metabolismo , Macrófagos/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Oligoelementos/efectos adversos , Células Tumorales CultivadasRESUMEN
Metal-on-metal (MoM) hip replacements, often manufactured from a cobalt-chrome alloy, are associated with adverse reactions including soft tissue necrosis and osteolysis. Histopathological analysis of MoM peri-implant tissues reveals an inflammatory cell infiltrate that includes macrophages, monocytes and neutrophils. Toll-like receptor 4 (TLR4) is an innate immune receptor activated by bacterial lipopolysaccharide. Recent studies have demonstrated that cobalt ions from metal-on-metal joints also activate human TLR4, increasing cellular secretion of inflammatory chemokines including interleukin-8 (IL-8, CXCL8) and CCL2. Chemokines recruit immune cells to the site of inflammation, and their overall effect depends on the chemokine profile produced. This study investigated the effect of cobalt on the secretion of inflammatory cytokines CCL20 and IL-6. The chemotactic potential of conditioned media from a cobalt-stimulated human monocyte cell line on primary monocytes and neutrophils was investigated using an in vitro transwell migration assay. The role of TLR4 in observed effects was studied using a small molecule TLR4-specific antagonist. Cobalt ions significantly increased release of CCL2 and IL-6 by MonoMac 6 cells (P<0.001). Conditioned media from cobalt-stimulated cells significantly increased monocyte and neutrophil chemotaxis in vitro (P<0.001). These effects were abrogated by the TLR4 antagonist (P<0.001) suggesting that they occur through cobalt activation of TLR4. This study demonstrates the role of TLR4 in cobalt-mediated immune cell chemotaxis and provides a potential mechanism by which cobalt ions may contribute to the immune cell infiltrate surrounding failed metal hip replacements. It also highlights the TLR4 signalling pathway as a potential therapeutic target in preventing cobalt-mediated inflammation.
RESUMEN
Overexpression of the oncogene HER2 occurs in 20-30% of invasive breast cancer and is associated with poor prognosis. A number of different splice variants of HER2 have been identified which produce functionally different proteins. Previously these splice variants have been investigated separately, but in the present study we collectively look at the expression and regulation of a group of HER2 splice variants produced by a splicing hotspot. Initial investigation in a cohort of tumor samples showed large variations in HER2 variant expression between patient samples. RNA interference studies identified 2 splicing factors involved in the regulation of splicing within this region, hnRNP H1 and SRSF3. siRNA targeting hnRNP H1 increases levels of X5 and the oncogenic variant Δ16HER2. Furthermore RNA chromatography assays demonstrated binding of hnRNP H1 to RNA in this region. Additionally the proto-oncogene SRSF3 was also identified as an important regulator of splicing with SRSF3 knockdown resulting in changes in all the splice variants located at the hotspot. Most notably knockdown of SRSF3 resulted in a switch from the oncogenic Δ16HER2 to p100 which inhibits cell proliferation. Binding of SRSF3 to RNA within this region was also demonstrated by RNA chromatography and more specifically 2 SRSF3 binding sites were identified within exon 15. SRSF3 and hnRNP H1 are the first splicing factors identified which regulate the production of these functionally distinct HER2 splice variants and therefore maybe important for the regulation of HER2 signaling.
Asunto(s)
Empalme Alternativo/genética , Neoplasias de la Mama/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Proteínas de Unión al ARN/genética , Receptor ErbB-2/genética , Sitios de Unión , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Humanos , Proto-Oncogenes Mas , Proteínas de Unión al ARN/metabolismo , Receptor ErbB-2/metabolismo , Factores de Empalme Serina-ArgininaRESUMEN
Scaffold attachment factor B1 (SAFB1) and SAFB2 proteins are oestrogen (ER) corepressors that bind to and modulate ER activity through chromatin remodelling or interaction with the basal transcription machinery. SAFB proteins also have an internal RNA-recognition motif but little is known about the RNA-binding properties of SAFB1 or SAFB2. We utilised crosslinking and immunoprecipitation (iCLIP) coupled with high-throughput sequencing to enable a transcriptome-wide mapping of SAFB1 protein-RNA interactions in breast cancer MCF-7 cells. Analysis of crosslinking frequency mapped to transcript regions revealed that SAFB1 binds to coding and noncoding RNAs (ncRNAs). The highest proportion of SAFB1 crosslink sites mapped to ncRNAs, followed by intergenic regions, open reading frames (ORFs), introns, and 3' or 5' untranslated regions (UTR). Furthermore, we reveal that SAFB1 binds directly to RNA and its binding is particularly enriched at purine-rich sequences not dissimilar to the RNA-binding motifs for SR proteins. Using RNAi, we also show, for the first time, that single depletion of either SAFB1 or SAFB2 leads to an increase in expression of the other SAFB protein in both MCF-7 and MDA-MD231 breast cancer cells.
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Neoplasias de la Mama/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas de Unión al ARN/genética , ARN/genética , Receptores de Estrógenos/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Línea Celular Tumoral , Estrógenos/genética , Femenino , Humanos , Intrones/genética , Células MCF-7 , Sistemas de Lectura Abierta/genética , Interferencia de ARN/fisiologíaRESUMEN
It is well known that many genes implicated in the development and progression of breast cancer undergo aberrant alternative splicing events to produce proteins with pro-cancer properties. These changes in alternative splicing can arise from mutations or single-nucleotide polymorphisms (SNPs) within the DNA sequences of cancer-related genes, which can strongly affect the activity of splicing factors and influence the splice site choice. However, it is important to note that absence of mutations is not sufficient to prevent misleading choices in splice site selection. There is now increasing evidence to demonstrate that the expression profile of ten splicing factors (including SRs and hnRNPs) and eight RNA-binding proteins changes in breast cancer cells compared with normal cells. These modifications strongly influence the alternative splicing pattern of many cancer-related genes despite the absence of any detrimental mutations within their DNA sequences. Thus, a comprehensive assessment of the splicing factor status in breast cancer is important to provide insights into the mechanisms that lead to breast cancer development and metastasis. Whilst most studies focus on mutations that affect alternative splicing in cancer-related genes, this review focuses on splicing factors and RNA-binding proteins that are themselves deregulated in breast cancer and implicated in cancer-related alternative splicing events.
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Empalme Alternativo/fisiología , Neoplasias de la Mama/metabolismo , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo/genética , Animales , Neoplasias de la Mama/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Proteínas de Unión al ARN/genéticaRESUMEN
Histological chorioamnionitis (HCA) is an established marker of ascending infection, a major cause of preterm birth. No studies have characterised the global change in expression of genes involved in the toll-like receptor (TLR) signalling pathways in the presence of HCA in the setting of preterm birth (pHCA). Fetal membranes were collected immediately after delivery and underwent histological staging for inflammation to derive 3 groups; term spontaneous labour without HCA (n = 9), preterm birth <34 weeks gestation without HCA (n = 8) and pHCA <34 weeks (n = 12). Profiling arrays ran in triplicate for each group were used to determine the expression of 84 genes associated with TLR signalling and screen for genes of interest (fold change >2; p<0.1). Expression of identified genes was validated individually for all samples, relative to GAPDH, using RT-PCR. Expression of TLR 1, TLR 2, lymphocyte antigen 96, interleukin 8 and Interleukin-1 receptor-associated kinase-like 2 was increased in pHCA (p<0.05). Degree of expression was positively associated with histological staging of both maternal and fetal inflammation (p<0.05). The inflammatory expression profile at the maternal/fetal interface associated with pHCA, a reflection of ascending infection, is extremely heterogeneous suggesting polymicrobial involvement with activation of a common pathway. Antagonism of TLR 1 and TLR 2 signalling in this setting warrants further assessment.
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Amnios/metabolismo , Corioamnionitis/metabolismo , Nacimiento Prematuro/metabolismo , Transducción de Señal , Receptor Toll-Like 1/metabolismo , Adulto , Corioamnionitis/diagnóstico , Femenino , Humanos , Embarazo , Nacimiento Prematuro/diagnóstico , Receptor Toll-Like 1/genética , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Regulación hacia ArribaRESUMEN
Alternative splicing--the production of multiple messenger RNA isoforms from a single gene--is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2ß have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous--but not individual--depletion of Tra2α and Tra2ß induces substantial shifts in splicing of endogenous Tra2ß target exons, and that both constitutive and alternative target exons are under dual Tra2α-Tra2ß control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability.
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Empalme Alternativo , Exones , Proteínas del Tejido Nervioso/metabolismo , Proteínas Quinasas/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Humanos , Células MCF-7 , Proteínas Quinasas/metabolismo , Factores de Empalme Serina-ArgininaRESUMEN
Tra2 proteins regulate pre-mRNA splicing in vertebrates and invertebrates, and are involved in important processes ranging from brain development in mice to sex determination in fruitflies. In structure Tra2 proteins contain two RS domains (domains enriched in arginine and serine residues) flanking a central RRM (RNA recognition motif). Understanding the mechanisms of how Tra2 proteins work to control splicing is one of the key requirements to understand their biology. In the present article, we review what is known about how Tra2 proteins regulate splicing decisions in mammals and fruitflies.
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Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Empalme Alternativo , Animales , DrosophilaRESUMEN
The majority of deaths in ovarian cancer are caused by recurrent metastatic disease which is usually multidrug resistant. This progression has been hypothesised to be due in part to the presence of cancer stem cells, a subset of cells which are capable of self-renewal and are able to survive chemotherapy and migrate to distant sites. Side population (SP) cells, identified by the efflux of the DNA-binding dye Hoechst 33342 through ATP-binding cassette (ABC) transporters, are a known adult stem cell group and have been suggested as a cancer stem cell in various cancers. Despite the identification of SP cells in cancer cell lines and patient samples, little attention has been paid to the identification of specific ABC transporters within this cell fraction which efflux Hoechst dye and thus may facilitate drug resistance. In this study, we demonstrate that SP cells can be detected in both ovarian cancer cell lines and ascitic fluid samples, and these SP cells possess stem cell and drug resistance properties. We show that ABCB1 is the functioning ABC transporter in ovarian cancer cell lines, and expression of ABCB1 is associated with a paclitaxel-resistant phenotype. Moreover, silencing of ABCB1 using a specific morpholino oligonucleotide results in an inhibition of the SP phenotype and a sensitising of ovarian cancer cell lines to paclitaxel. ABCB1 should therefore be considered as a therapeutic target in ovarian cancer.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/metabolismo , Células de Población Lateral/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antineoplásicos Fitogénicos/farmacología , Western Blotting , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Ováricas/patología , Paclitaxel/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The splicing regulator proteins SRSF1 (also known as ASF/SF2) and SRSF3 (also known as SRP20) belong to the SR family of proteins and can be upregulated in cancer. The SRSF1 gene itself is amplified in some cancer cells, and cancer-associated changes in the expression of MYC also increase SRSF1 gene expression. Increased concentrations of SRSF1 protein promote prooncogenic splicing patterns of a number of key regulators of cell growth. Here, we review the evidence that upregulation of the SR-related Tra2 ß protein might have a similar role in cancer cells. The TRA2B gene encoding Tra2 ß is amplified in particular tumours including those of the lung, ovary, cervix, stomach, head, and neck. Both TRA2B RNA and Tra2 ß protein levels are upregulated in breast, cervical, ovarian, and colon cancer, and Tra2 ß expression is associated with cancer cell survival. The TRA2B gene is a transcriptional target of the protooncogene ETS-1 which might cause higher levels of expression in some cancer cells which express this transcription factor. Known Tra2 ß splicing targets have important roles in cancer cells, where they affect metastasis, proliferation, and cell survival. Tra2 ß protein is also known to interact directly with the RBMY protein which is implicated in liver cancer.