RESUMEN
A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis.
Asunto(s)
Glucolípidos , Síndrome de Guillain-Barré , Mycoplasma pneumoniae , Síndrome de Guillain-Barré/microbiología , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/inmunología , Humanos , Glucolípidos/metabolismo , Galactosilceramidas , Reacciones Cruzadas , Neumonía por Mycoplasma/microbiología , Neumonía por Mycoplasma/inmunología , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunologíaRESUMEN
Identifying open reading frames (ORFs) being translated is not a trivial task. ProTInSeq is a technique designed to characterize proteomes by sequencing transposon insertions engineered to express a selection marker when they occur in-frame within a protein-coding gene. In the bacterium Mycoplasma pneumoniae, ProTInSeq identifies 83% of its annotated proteins, along with 5 proteins and 153 small ORF-encoded proteins (SEPs; ≤100 aa) that were not previously annotated. Moreover, ProTInSeq can be utilized for detecting translational noise, as well as for relative quantification and transmembrane topology estimation of fitness and non-essential proteins. By integrating various identification approaches, the number of initially annotated SEPs in this bacterium increases from 27 to 329, with a quarter of them predicted to possess antimicrobial potential. Herein, we describe a methodology complementary to Ribo-Seq and mass spectroscopy that can identify SEPs while providing other insights in a proteome with a flexible and cost-effective DNA ultra-deep sequencing approach.
Asunto(s)
Bacterias , Proteoma , Sistemas de Lectura Abierta/genética , Secuencia de Bases , Bacterias/genética , Proteoma/genética , Análisis de Secuencia de ADN , ADNRESUMEN
Snail1 is a transcriptional factor required for cancer-associated fibroblast (CAF) activation, and mainly detected in CAFs in human tumors. In the mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) model of murine mammary gland tumors, Snai1 gene deletion, besides increasing tumor-free lifespan, altered macrophage differentiation, with fewer expressing low levels of MHC class II. Snail1 was not expressed in macrophages, and in vitro polarization with interleukin-4 (IL4) or interferon-γ (IFNγ) was not altered by Snai1 gene depletion. We verified that CAF activation modified polarization of naïve bone-marrow-derived macrophages (BMDMΦs). When BMDMΦs were incubated with Snail1-expressing (active) CAFs or with conditioned medium derived from these cells, they exhibited a lower cytotoxic capability than when incubated with Snail1-deleted (inactive) CAFs. Gene expression analysis of BMDMΦs polarized by conditioned medium from wild-type or Snai1-deleted CAFs revealed that active CAFs differentially stimulated a complex combination of genes comprising genes that are normally induced by IL4, downregulated by IFNγ, or not altered during the two canonical differentiations. Levels of RNAs relating to this CAF-induced alternative polarization were sensitive to inhibitors of factors specifically released by active CAFs, such as prostaglandin E2 and TGFß. Finally, CAF-polarized macrophages promoted the activation of the immunosuppressive regulatory T cells (T-regs). Our results imply that an active CAF-rich tumor microenvironment induces the polarization of macrophages to an immunosuppressive phenotype, preventing the macrophage cytotoxic activity on tumor cells and enhancing the activation of T-reg cells.
Asunto(s)
Fibroblastos Asociados al Cáncer , Neoplasias , Humanos , Ratones , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Interleucina-4/farmacología , Medios de Cultivo Condicionados/metabolismo , Diferenciación Celular , Macrófagos/metabolismo , Microambiente Tumoral , Línea Celular Tumoral , Neoplasias/patologíaRESUMEN
Engineered live bacteria could provide a new modality for treating lung infections, a major cause of mortality worldwide. In the present study, we engineered a genome-reduced human lung bacterium, Mycoplasma pneumoniae, to treat ventilator-associated pneumonia, a disease with high hospital mortality when associated with Pseudomonas aeruginosa biofilms. After validating the biosafety of an attenuated M. pneumoniae chassis in mice, we introduced four transgenes into the chromosome by transposition to implement bactericidal and biofilm degradation activities. We show that this engineered strain has high efficacy against an acute P. aeruginosa lung infection in a mouse model. In addition, we demonstrated that the engineered strain could dissolve biofilms formed in endotracheal tubes of patients with ventilator-associated pneumonia and be combined with antibiotics targeting the peptidoglycan layer to increase efficacy against Gram-positive and Gram-negative bacteria. We expect our M. pneumoniae-engineered strain to be able to treat biofilm-associated infections in the respiratory tract.
Asunto(s)
Neumonía Asociada al Ventilador , Infecciones por Pseudomonas , Humanos , Ratones , Animales , Antibacterianos/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Neumonía Asociada al Ventilador/tratamiento farmacológico , Neumonía Asociada al Ventilador/microbiología , Bacterias Gramnegativas , Bacterias Grampositivas , Intubación Intratraqueal , Biopelículas , PulmónRESUMEN
Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.
Asunto(s)
Adhesinas Bacterianas/inmunología , Adhesión Bacteriana/inmunología , Mycoplasma pneumoniae/inmunología , Neumonía por Mycoplasma/inmunología , Adhesinas Bacterianas/aislamiento & purificación , Adhesinas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Mycoplasma pneumoniae/aislamiento & purificación , Mycoplasma pneumoniae/patogenicidad , Neumonía por Mycoplasma/sangre , Neumonía por Mycoplasma/microbiología , Dominios Proteicos/inmunologíaRESUMEN
Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of ß-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II' (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.
Asunto(s)
Alginatos/metabolismo , Proteínas Bacterianas/uso terapéutico , Terapia Biológica/métodos , Liasas/uso terapéutico , Infecciones por Pseudomonas/terapia , Pseudomonas aeruginosa/fisiología , Sphingobacterium/metabolismo , Antibacterianos/uso terapéutico , Proteínas Bacterianas/metabolismo , Biopelículas , Clonación Molecular , Mezclas Complejas , Sinergismo Farmacológico , Humanos , Liasas/metabolismo , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
Identification of small open reading frames (smORFs) encoding small proteins (≤ 100 amino acids; SEPs) is a challenge in the fields of genome annotation and protein discovery. Here, by combining a novel bioinformatics tool (RanSEPs) with "-omics" approaches, we were able to describe 109 bacterial small ORFomes. Predictions were first validated by performing an exhaustive search of SEPs present in Mycoplasma pneumoniae proteome via mass spectrometry, which illustrated the limitations of shotgun approaches. Then, RanSEPs predictions were validated and compared with other tools using proteomic datasets from different bacterial species and SEPs from the literature. We found that up to 16 ± 9% of proteins in an organism could be classified as SEPs. Integration of RanSEPs predictions with transcriptomics data showed that some annotated non-coding RNAs could in fact encode for SEPs. A functional study of SEPs highlighted an enrichment in the membrane, translation, metabolism, and nucleotide-binding categories. Additionally, 9.7% of the SEPs included a N-terminus predicted signal peptide. We envision RanSEPs as a tool to unmask the hidden universe of small bacterial proteins.
Asunto(s)
Proteínas Bacterianas/genética , Biología Computacional , Péptidos/genética , Proteoma/genética , Proteínas Bacterianas/aislamiento & purificación , Genoma Bacteriano/genética , Espectrometría de Masas , Mycoplasma/genética , ProteómicaRESUMEN
Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFß-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes.
Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción de la Familia Snail/genética , Homeostasis del Telómero/genética , Telómero/genética , Animales , Línea Celular , Células Cultivadas , Transición Epitelial-Mesenquimal/genética , Perfilación de la Expresión Génica/métodos , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones Noqueados , Células Madre Neoplásicas/metabolismo , ARN Largo no Codificante/genética , Factores de Transcripción de la Familia Snail/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Telómero/enzimologíaRESUMEN
Although deregulation of EPHB signaling has been shown to be an important step in colorectal tumorigenesis, the role of EPHB6 in this process has not been investigated. We found here that manipulation of EPHB6 levels in colon cancer cell lines has no effect on their motility and growth on a solid substrate, soft agar or in a xenograft mouse model. We then used an EphB6 knockout mouse model to show that EphB6 inactivation does not efficiently initiate tumorigenesis in the intestinal tract. In addition, when intestinal tumors are initiated genetically or pharmacologically in EphB6+/+ and EphB6-/- mice, no differences were observed in animal survival, tumor multiplicity, size or histology, and proliferation of intestinal epithelial cells or tumor cells. However, reintroduction of EPHB6 into colon cancer cells significantly reduced the number of lung metastasis after tail-vein injection in immunodeficient mice, while EPHB6 knockdown in EPHB6-expressing cells increased their metastatic spread. Consistently, although EPHB6 protein expression in a series of 130 primary colorectal tumors was not associated with patient survival, EPHB6 expression was significantly lower in lymph node metastases compared to primary tumors. Our results indicate that the loss of EPHB6 contributes to the metastatic process of colorectal cancer.
Asunto(s)
Biomarcadores de Tumor , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Receptores de la Familia Eph/deficiencia , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/mortalidad , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismoRESUMEN
In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.
Asunto(s)
Regulación de la Expresión Génica , Factor de Unión 1 al Potenciador Linfoide/genética , Empalme del ARN , ARN sin Sentido/metabolismo , Línea Celular , Células Epiteliales/metabolismo , Humanos , Factor de Unión 1 al Potenciador Linfoide/biosíntesis , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Factores de Transcripción COUP/metabolismo , Diferenciación Celular , Interleucina-17/metabolismo , Proteómica/métodos , Factores de Transcripción/metabolismo , Células 3T3-L1 , Adipogénesis , Animales , Extractos Celulares , Núcleo Celular/metabolismo , Regulación hacia Abajo , Células Madre Mesenquimatosas/metabolismo , Ratones , Modelos Biológicos , Proteínas Represoras , Reproducibilidad de los Resultados , Transducción de Señal , Factores de Transcripción de la Familia Snail , TransfecciónRESUMEN
Activation of the small GTPase RHOA has strong oncogenic effects in many tumour types, although its role in colorectal cancer remains unclear. Here we show that RHOA inactivation contributes to colorectal cancer progression/metastasis, largely through the activation of Wnt/ß-catenin signalling. RhoA inactivation in the murine intestine accelerates the tumorigenic process and in human colon cancer cells leads to the redistribution of ß-catenin from the membrane to the nucleus and enhanced Wnt/ß-catenin signalling, resulting in increased proliferation, invasion and de-differentiation. In mice, RHOA inactivation contributes to colon cancer metastasis and reduced RHOA levels were observed at metastatic sites compared with primary human colon tumours. Therefore, we have identified a new mechanism of activation of Wnt/ß-catenin signalling and characterized the role of RHOA as a novel tumour suppressor in colorectal cancer. These results constitute a shift from the current paradigm and demonstrate that RHO GTPases can suppress tumour progression and metastasis.
Asunto(s)
Neoplasias del Colon/enzimología , Silenciador del Gen , Transducción de Señal , Proteínas Wnt/metabolismo , Proteína de Unión al GTP rhoA/genética , Animales , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Snail1 transcriptional repressor is a major inducer of epithelial-to mesenchymal transition but is very limitedly expressed in adult animals. We have previously demonstrated that Snail1 is required for the maintenance of mesenchymal stem cells (MSCs), preventing their premature differentiation. Now, we show that Snail1 controls the tumorigenic properties of mesenchymal cells. Increased Snail1 expression provides tumorigenic capabilities to fibroblastic cells; on the contrary, Snail1 depletion decreases tumor growth. Genetic depletion of Snail1 in MSCs that are deficient in p53 tumor suppressor downregulates MSC markers and prevents the capability of these cells to originate sarcomas in immunodeficient SCID mice. Notably, an analysis of human sarcomas shows that, contrarily to epithelial tumors, these neoplasms display high Snail1 expression. This is particularly clear for undifferentiated tumors, which are associated with poor outcome. Together, our results indicate a role for Snail1 in the generation of sarcomas.
Asunto(s)
Carcinogénesis/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Sarcoma/metabolismo , Factores de Transcripción/biosíntesis , Animales , Western Blotting , Carcinogénesis/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Estimación de Kaplan-Meier , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Ratones SCID , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma/genética , Sarcoma/mortalidad , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genéticaRESUMEN
Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MSI gastric and endometrial tumors and found a high mutation rate in gastric (22/47; 46.8%) but not endometrial (3/48; 6.2%) tumors. Using a regression model, we show that MYO1A mutations are likely to confer a growth advantage to gastric, but not endometrial tumors. The mutant MYO1A(7A) protein was shown to lose its membrane localization in gastric cancer cells and a cycloheximide-chase assay demonstrated that the mutant MYO1A(7A) protein has reduced stability compared to the wild type MYO1A. Frequent MYO1A promoter hypermethylation was also found in gastric tumors. Promoter methylation negatively correlates with MYO1A mRNA expression in a series of 58 non-MSI gastric primary tumors (Pearson's r = -0.46; p = 0.0003) but not in a cohort of 54 non-MSI endometrial tumors and treatment of gastric cancer cells showing high MYO1A promoter methylation with the demethylating agent 5-aza-2'-deoxycytidine, resulted in a significant increase of MYO1A mRNA levels. We found that normal gastric epithelial cells, but not normal endometrial cells, express high levels of MYO1A. Therefore, when considered together, our findings suggest that MYO1A has tumor suppressor activity in the normal gastric epithelium but not in the normal endometrium and inactivation of MYO1A either genetically or epigenetically may confer gastric epithelial cells a growth advantage.
Asunto(s)
Neoplasias Endometriales/genética , Microvellosidades/metabolismo , Cadenas Pesadas de Miosina/genética , Miosina Tipo I/genética , Neoplasias Gástricas/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Western Blotting , Metilación de ADN , Cartilla de ADN , Decitabina , Neoplasias Endometriales/patología , Femenino , Humanos , Microscopía Confocal , Mutación , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/patologíaRESUMEN
Colorectal cancers (CRCs) are classified as having microsatellite instability (MSI) or chromosomal instability (CIN); herein termed microsatellite stable (MSS). MSI colon cancers frequently display a poorly differentiated histology for which the molecular basis is not well understood. Gene expression and immunohistochemical profiling of MSS and MSI CRC cell lines and tumors revealed significant down-regulation of the intestinal-specific cytoskeletal protein villin in MSI colon cancer, with complete absence in 62% and 17% of MSI cell lines and tumors, respectively. Investigation of 577 CRCs linked loss of villin expression to poorly differentiated histology in MSI and MSS tumors. Furthermore, mislocalization of villin from the membrane was prognostic for poorer outcome in MSS patients. Loss of villin expression was not due to coding sequence mutations, epigenetic inactivation, or promoter mutation. Conversely, in transient transfection assays villin promoter activity reflected endogenous villin expression, suggesting transcriptional control. A screen of gut-specific transcription factors revealed a significant correlation between expression of villin and the homeobox transcription factor Cdx-1. Cdx-1 overexpression induced villin promoter activity, Cdx-1 knockdown down-regulated endogenous villin expression, and deletion of a key Cdx-binding site within the villin promoter attenuated promoter activity. Loss of Cdx-1 expression in CRC lines was associated with Cdx-1 promoter methylation. These findings demonstrate that loss of villin expression due to Cdx-1 loss is a feature of poorly differentiated CRCs.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Biomarcadores de Tumor/genética , Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Metilación de ADN/genética , ADN de Neoplasias/genética , Regulación hacia Abajo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones SCID , Proteínas de Microfilamentos/genética , Inestabilidad de Microsatélites , Repeticiones de Microsatélite , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Pronóstico , Regiones Promotoras Genéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Células Tumorales CultivadasRESUMEN
The loss of the epithelial architecture and cell polarity/differentiation is known to be important during the tumorigenic process. Here we demonstrate that the brush border protein Myosin Ia (MYO1A) is important for polarization and differentiation of colon cancer cells and is frequently inactivated in colorectal tumors by genetic and epigenetic mechanisms. MYO1A frame-shift mutations were observed in 32% (37 of 116) of the colorectal tumors with microsatellite instability analyzed, and evidence of promoter methylation was observed in a significant proportion of colon cancer cell lines and primary colorectal tumors. The loss of polarization/differentiation resulting from MYO1A inactivation is associated with higher tumor growth in soft agar and in a xenograft model. In addition, the progression of genetically and carcinogen-initiated intestinal tumors was significantly accelerated in Myo1a knockout mice compared with Myo1a wild-type animals. Moreover, MYO1A tumor expression was found to be an independent prognostic factor for colorectal cancer patients. Patients with low MYO1A tumor protein levels had significantly shorter disease-free and overall survival compared with patients with high tumoral MYO1A (logrank test P = 0.004 and P = 0.009, respectively). The median time-to-disease recurrence in patients with low MYO1A was 1 y, compared with >9 y in the group of patients with high MYO1A. These results identify MYO1A as a unique tumor-suppressor gene in colorectal cancer and demonstrate that the loss of structural brush border proteins involved in cell polarity are important for tumor development.
Asunto(s)
Genes Supresores de Tumor , Mucosa Intestinal/metabolismo , Microvellosidades/metabolismo , Miosina Tipo I/fisiología , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Metilación de ADN , Humanos , Mutación , Miosina Tipo I/genética , Regiones Promotoras GenéticasRESUMEN
PURPOSE: Irinotecan (CPT11) treatment significantly improves the survival of colorectal cancer patients and is routinely used for the treatment of these patients, alone or in combination with other agents. However, only 20% to 30% of patients show an objective response to irinotecan, and there is great need for new molecular markers capable of identifying the subset of patients who are unlikely to respond. EXPERIMENTAL DESIGN: Here we used microarray analysis of a panel of 30 colorectal cancer cell lines and immunohistochemistry to identify and validate a new biomarker of response to irinotecan. RESULTS: A good correlation was observed between irinotecan sensitivity and the expression of aprataxin (APTX), a histidine triad domain superfamily protein involved in DNA repair. Moreover, using an isogenic in vitro system deficient in APTX, we show that aprataxin directly regulates the cellular sensitivity to camptothecin, suggesting that it could be used to predict patient response to irinotecan. We constructed a tissue microarray containing duplicate tumor samples from 135 patients that received irinotecan for the treatment of advanced colorectal cancer. Immunohistochemical assessment of the tumor levels of aprataxin showed a significant association with treatment response and patient survival. Patients with low aprataxin had longer progression-free (9.2 versus 5.5 months; P = 0.03) and overall survival (36.7 versus 19.0 months; P = 0.008) than patients with high tumor aprataxin. No associations were found between coding APTX variants and aprataxin levels or camptothecin sensitivity. CONCLUSIONS: These results show that aprataxin tumor levels can be used to identify patients with low probability of response to irinotecan-based therapy who are ideal candidates to receive treatment with alternative agents in an attempt to improve patient survival.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Camptotecina/análogos & derivados , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Camptotecina/uso terapéutico , Proliferación Celular , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Irinotecán , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo Genético , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Análisis de Matrices Tisulares , Inhibidores de Topoisomerasa I , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
Colorectal cancer is the second cause of cancer-related death in the western world, and although the genetic and molecular mechanisms involved in the initiation and progression of these tumors are among the best characterized, there are significant gaps in our understanding of this disease. The role of EPHB signaling in colorectal cancer has only recently been realized. Here, we use animal models to investigate the role of EphB4 in intestinal tumorigenesis. Modulation of EPHB4 levels in colon cancer cell lines resulted in significant differences in tumor growth in a xenograft model, with low levels of EPHB4 associated with faster growth. In addition, using a genetic model of intestinal tumorigenesis where adenomatous polyposis coli (Apc) mutations lead to initiation of the tumorigenic process (Apc(min) mice), we show that inactivation of a single allele of EphB4 results in higher proliferation in both the normal epithelium and intestinal tumors, significantly larger tumors in the small intestine, and a 10-fold increase in the number of tumors in the large intestine. This was associated with a 25% reduction in the lifespan of Apc(min) mice (P < 0.0001). Gene expression analysis showed that EphB4 mutations result in a profound transcriptional reprogramming, affecting genes involved in cell proliferation, remodeling of the extracellular matrix, and cell attachment to the basement membrane among other functional groups of genes. Importantly, in agreement with the expression profiling experiments, using an in vitro assay, we show that loss of EPHB4 in colon cancer cells results in a significantly increased potential to invade through a complex extracellular matrix. Collectively, these results indicate that EphB4 has tumor suppressor activities and that regulation of cell proliferation, extracellular matrix remodeling, and invasive potential are important mechanisms of tumor suppression.