RESUMEN
Down's syndrome (DS) is a genetic disorder caused by full or partial trisomy of human chromosome 21 and presents with many clinical phenotypes including a reduced incidence of solid tumours. Recent work with the Ts65Dn model of DS, which has orthologues of about 50% of the genes on chromosome 21 (Hsa21), has indicated that three copies of the ETS2 (ref. 3) or DS candidate region 1 (DSCR1) genes (a previously known suppressor of angiogenesis) is sufficient to inhibit tumour growth. Here we use the Tc1 transchromosomic mouse model of DS to dissect the contribution of extra copies of genes on Hsa21 to tumour angiogenesis. This mouse expresses roughly 81% of Hsa21 genes but not the human DSCR1 region. We transplanted B16F0 and Lewis lung carcinoma tumour cells into Tc1 mice and showed that growth of these tumours was substantially reduced compared with wild-type littermate controls. Furthermore, tumour angiogenesis was significantly repressed in Tc1 mice. In particular, in vitro and in vivo angiogenic responses to vascular endothelial growth factor (VEGF) were inhibited. Examination of the genes on the segment of Hsa21 in Tc1 mice identified putative anti-angiogenic genes (ADAMTS1and ERG) and novel endothelial cell-specific genes, never previously shown to be involved in angiogenesis (JAM-B and PTTG1IP), that, when overexpressed, are responsible for inhibiting angiogenic responses to VEGF. Three copies of these genes within the stromal compartment reduced tumour angiogenesis, explaining the reduced tumour growth in DS. Furthermore, we expect that, in addition to the candidate genes that we show to be involved in the repression of angiogenesis, the Tc1 mouse model of DS will permit the identification of other endothelium-specific anti-angiogenic targets relevant to a broad spectrum of cancer patients.
Asunto(s)
Carcinoma Pulmonar de Lewis/irrigación sanguínea , Modelos Animales de Enfermedad , Síndrome de Down/genética , Dosificación de Gen/genética , Melanoma Experimental/irrigación sanguínea , Neovascularización Patológica/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS1 , Animales , Carcinoma Pulmonar de Lewis/complicaciones , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cromosomas de los Mamíferos/genética , Síndrome de Down/complicaciones , Síndrome de Down/fisiopatología , Femenino , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Masculino , Melanoma Experimental/complicaciones , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Trasplante de Neoplasias , Neovascularización Patológica/patología , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteína Proto-Oncogénica c-ets-2/genética , Proteína Proto-Oncogénica c-ets-2/metabolismo , Factores de Transcripción , Regulador Transcripcional ERG , Trisomía/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death, largely due to metastatic disease. To better understand PDAC metastatic spread and identify novel therapeutic targets, we analysed the proteome of primary tumours and matched lymph node (LN) metastases. As frozen specimens of metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded (FFPE) tissues. This poses technical challenges because of the cross-linkages induced by fixation. Using laser capture microdissection (PALM system), we isolated malignant epithelia from seven FFPE primary PDAC tumours and matched LN metastases. Following dissection, samples were analysed in duplicate using Multidimensional Protein Identification Technology (MudPIT); this resulted in the identification of 1504 proteins, 854 of which were common to all samples analysed. Comparison of the obtained proteins with data from previous proteomics studies on pancreatic tissue, pancreatic juice, serum, and urine resulted in a less than 30% overlap, indicating that our study has substantially expanded the current database of proteins expressed in this malignancy. Statistical analysis further showed that 115/854 proteins (13.5%) were significantly differentially expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly significant g-values were confirmed to be significantly differentially expressed (S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) in a larger series of 55 cases of matched primary PDAC and LN metastases using immunohistochemistry. Thus, laser capture microdissection of FFPE tissue coupled with downstream proteomic analysis is a valid approach for the investigation of metastatic PDAC. This is the first study to establish and compare the protein composition of primary PDAC and matched LN metastases, and has resulted in the identification of several potential epithelial-specific therapeutic targets, including 14-3-3 sigma and S100P.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Ductal Pancreático/química , Fijadores , Formaldehído , Ganglios Linfáticos/química , Proteínas de Neoplasias/análisis , Neoplasias Pancreáticas/química , Adhesión en Parafina , Proteómica , Fijación del Tejido/métodos , Proteínas 14-3-3/análisis , Proteínas de Unión al Calcio/análisis , Carcinoma Ductal Pancreático/secundario , Exonucleasas/análisis , Exorribonucleasas , Humanos , Inmunohistoquímica , Captura por Microdisección con Láser , Ganglios Linfáticos/patología , Metástasis Linfática , Neoplasias Pancreáticas/patología , Pronóstico , Proteómica/métodos , Espectrometría de Masas en TándemRESUMEN
E-cadherin, a classical cadherin, is an adhesion receptor in adherens junctions and has important functions in cell-cell adhesion and cell signalling. Recently we reported that a desmosomal cadherin, desmoglein 3 (Dsg3), an autoantigen in pemphigus vulgaris (PV), associates with E-cadherin and activates Src, which results in tyrosine phosphorylation of adherens junction proteins. However, the nature of such an interaction and its role in cell-cell adhesion remain unclear. In this report, we provide direct evidence that it is the detergent-soluble, non-desmosomal Dsg3 that regulates the activity of Src and its association with E-cadherin in adherens junction formation. Modulation of Dsg3 levels, either by Dsg3 silencing or over-expression, alters Src activity and its association with E-cadherin. Dsg3 silencing caused retardation of calcium-induced E-cadherin junction assembly and a reduction of desmosomal protein expression. Furthermore, we provide evidence that this signalling pathway is involved, at least in part, in the pathophysiology of pemphigus. Along with the reduced expression of Dsg3, loss and disruption of E-cadherin and a concomitant decreased pSrc signalling was identified in the basal keratinocytes surrounding the blisters in PV. These findings suggest a novel function for Dsg3 in the control of E-cadherin-Src signalling and cell-cell adhesion.
Asunto(s)
Cadherinas/metabolismo , Desmogleína 3/genética , Regulación de la Expresión Génica , Pénfigo/genética , Proteínas Tirosina Quinasas/genética , Proteína Tirosina Quinasa CSK , Adhesión Celular/genética , Línea Celular , Desmogleína 3/metabolismo , Activación Enzimática , Silenciador del Gen , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Mucosa Bucal/metabolismo , Mucosa Bucal/patología , Pénfigo/metabolismo , Pénfigo/patología , Proteínas Tirosina Quinasas/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transducción de Señal , Transfección , Familia-src QuinasasRESUMEN
BACKGROUND: The mammalian inner ear contains the organ of Corti which is responsible for the conversion of sound into neuronal signals. This specialised epithelial tissue is the product of a complex developmental process where a common precursor cell type differentiates into the sound transducing hair cells and the non-innervated supporting cells. We hypothesised that integrin proteins, which are involved in cell attachment to extracellular matrix proteins and cellular signalling, play a role in the differentiation process of the precursor inner ear epithelial cells. To test our hypothesis we have utilised a cell line (OC-2) derived from E13 embryonic immortomouse inner ears. In vitro, by switching the incubation temperature from 33°C to 39°C, the OC-2 cells can be induced to differentiate and express hair cells markers, such as Myosin VIIa. The OC-2 cells are thus a useful model system for testing mechanism of hair cells differentiation. RESULTS: We have identified 4 integrin subunits which are expressed in OC-2 cells: α6, αv, ß1 and ß3. Among these, the relative level of expression of the αv, ß1 and ß3 subunits increased in a time dependent manner when the cells were exposed to the differentiating temperature of 39°C, most notably so for ß3 which was not detectable at 33°C. Treatment of fully differentiated OC-2 cells with siRNA against the four integrin subunits reduced the expression of not only the respective integrin proteins but also of the hair cell marker Myosin VIIa. Conversely over-expression of ß3 was sufficient to induce the expression of Myosin VIIa at 33°C. CONCLUSIONS: Our data demonstrate that modulation of integrin expression is associated with the differentiation process of the OC-2 cells. This suggests that the maturation of the organ of Corti, from where OC-2 cells are derived, may also depend on changes of gene expression associated with integrin expression.
Asunto(s)
Diferenciación Celular , Embrión de Mamíferos/citología , Integrina beta3/metabolismo , Órgano Espiral/citología , Órgano Espiral/metabolismo , Animales , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Integrina beta3/genética , Ratones , Órgano Espiral/embriologíaRESUMEN
BACKGROUND & AIMS: Patients with pancreatic ductal adenocarcinoma are deficient in vitamin A, resulting in activation of pancreatic stellate cells (PSCs). We investigated whether restoration of retinol to PSCs restores their quiescence and affects adjacent cancer cells. METHODS: PSCs and cancer cell lines (AsPc1 and Capan1) were exposed to doses and isoforms of retinoic acid (RA) in 2-dimensional and 3-dimensional culture conditions (physiomimetic organotypic culture). The effects of all-trans retinoic acid (ATRA) were studied in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice, a model of human pancreatic ductal adenocarcinoma. RESULTS: After incubation with ATRA, PSCs were quiescent and had altered expression of genes that regulate proliferation, morphology, and motility; genes that encode cytoskeletal proteins and cytokines; and genes that control other functions, irrespective of culture conditions or dosage. In the organotypic model, and in mice, ATRA induced quiescence of PSCs and thereby reduced cancer cell proliferation and translocation of ß-catenin to the nucleus, increased cancer cell apoptosis, and altered tumor morphology. ATRA reduced the motility of PSCs, so these cells created a "wall" at the junction between the tumor and the matrix that prevented cancer cell invasion. Restoring secreted frizzled-related protein 4 (sFRP4) secretion to quiescent PSCs reduced Wnt-ß-catenin signaling in cancer cells and their invasive ability. Human primary and metastatic pancreatic tumor tissues stained strongly for cancer cell nuclear ß-catenin but had low levels of sFRP4 (in cancer cells and PSCs). CONCLUSIONS: RA induces quiescence and reduces motility of PSCs, leading to reduced proliferation and increased apoptosis of surrounding pancreatic cancer cells. RA isoforms might be developed as therapeutic reagents for pancreatic cancer.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Senescencia Celular/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Células Estrelladas Pancreáticas/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Alitretinoína , Animales , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isotretinoína/farmacología , Ratones , Ratones Mutantes , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Células Estrelladas Pancreáticas/metabolismo , Células Estrelladas Pancreáticas/patología , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Integrin α9ß1 is a receptor for ECM proteins, including Tenascin-C and the EDA domain of fibronectin, and has been shown to transduce TGFß signalling. This study has examined the expression pattern of α9ß1 in 141 frozen breast carcinoma samples and related expression to prognostic indices, molecular subtype and patient outcome. Effects of α9ß1 on tumour cell migration and invasion were assessed using blocking antibody and gene transduction approaches. Integrin α9ß1 localized to myoepithelial cells in normal ducts and acini, a pattern maintained in DCIS. A subset (17%) of invasive carcinomas exhibited tumour cell expression of α9ß1, which related significantly to the basal-like phenotype, as defined by either CK5/6 or CK14 expression. Tumour expression of α9ß1 showed a significant association with reduced overall patient survival (p < 0.0001; HR 5.94, 95%CI 3.26-10.82) and with reduced distant-metastasis-free survival (p < 0.0001; HR 6.37, CI 3.51-11.58). A series of breast cancer cell lines was screened for α9ß1 with the highly invasive basal-like GI-101 cell line expressing significant levels. Both migration and invasion of this line were reduced significantly in the presence of α9-blocking antibody and following α9-knockdown with siRNA. Conversely, migratory and invasive behaviour of α9-negative MCF7 cells and α9-low MDA MB468 cells was enhanced significantly by over-expression of α9. Thus, α9ß1 acts as a novel marker of the basal-like breast cancer subtype and expression is associated with reduced survival, while its ability to promote breast cancer cell migration and invasion suggests that it contributes to the aggressive clinical behaviour of this tumour subtype.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Integrinas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Proliferación Celular , Femenino , Humanos , Integrinas/fisiología , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Fenotipo , Pronóstico , Análisis de Supervivencia , Células Tumorales CultivadasRESUMEN
Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7-13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As αvß6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. αvß6 was markedly up-regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of αvß6 using oral keratinocyte-derived cells genetically modified to express high αvß6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low αvß6 (OKF6/TERT-1). VB6 cells showed significant αvß6-dependent activation of TGF-ß1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF-ß1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte αvß6 expression. This was modulated through the M(4) muscarinic acetylcholine receptor and was suppressed by the M(4) antagonist, tropicamide. Arecoline-dependent αvß6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF-related oral cancers examined had moderate/high αvß6 expression. These data suggest that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of αvß6 integrin.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Areca/química , Arecolina/farmacología , Integrinas/biosíntesis , Queratinocitos/efectos de los fármacos , Fibrosis de la Submucosa Bucal/metabolismo , Actinas/metabolismo , Antígenos de Neoplasias/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Humanos , Integrinas/genética , Queratinocitos/metabolismo , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Miofibroblastos/citología , Miofibroblastos/efectos de los fármacos , Fibrosis de la Submucosa Bucal/patología , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Worldwide, approximately 405 000 cases of oral cancer (OSCC) are diagnosed each year, with a rising incidence in many countries. Despite advances in surgery and radiotherapy, which remain the standard treatment options, the mortality rate has remained largely unchanged for decades, with a 5-year survival rate of around 50%. OSCC is a heterogeneous disease, staged currently using the TNM classification, supplemented with pathological information from the primary tumour and loco-regional lymph nodes. Although patients with advanced disease show reduced survival, there is no single pathological or molecular feature that identifies aggressive, early-stage tumours. We retrospectively analysed 282 OSCC patients for disease mortality, related to clinical, pathological, and molecular features based on our previous functional studies [EGFR, αvß6 integrin, smooth muscle actin (SMA), p53, p16, EP4]. We found that the strongest independent risk factor of early OSCC death was a feature of stroma rather than tumour cells. After adjusting for all factors, high stromal SMA expression, indicating myofibroblast transdifferentiation, produced the highest hazard ratio (3.06, 95% CI 1.65-5.66) and likelihood ratio (3.6; detection rate: false positive rate) of any feature examined, and was strongly associated with mortality, regardless of disease stage. Functional assays showed that OSCC cells can modulate myofibroblast transdifferentiation through αvß6-dependent TGF-ß1 activation and that myofibroblasts promote OSCC invasion. Finally, we developed a prognostic model using Cox regression with backward elimination; only SMA expression, metastasis, cohesion, and age were significant. This model was independently validated on a patient subset (detection rate 70%; false positive rate 20%; ROC analysis 77%, p < 0.001). Our study highlights the limited prognostic value of TNM staging and suggests that an SMA-positive, myofibroblastic stroma is the strongest predictor of OSCC mortality. Whether used independently or as part of a prognostic model, SMA identifies a significant group of patients with aggressive tumours, regardless of disease stage.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias de la Boca/patología , Células del Estroma/patología , Actinas/metabolismo , Anciano , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/secundario , Carcinoma de Células Escamosas/terapia , Métodos Epidemiológicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/terapia , Miofibroblastos/fisiología , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Estadificación de Neoplasias , Pronóstico , Células del Estroma/metabolismoRESUMEN
Effective reepithelialization after injury is essential for correct wound healing. The upregulation of keratinocyte alpha3beta1 integrin during reepithelialization suggests that this adhesion molecule is involved in wound healing; however, its precise role in this process is unknown. We have shown here that retarded reepithelialization in Itga3(-/-) mouse skin wounds is due predominantly to repressed TGF-beta1-mediated responses. Specifically, expression of the inhibitor of TGF-beta1-signaling Smad7 was elevated in Itga3(-/-) keratinocytes. Indeed, in vivo blockade of Smad7 increased the rate of reepithelialization in Itga3(-/-) and WT wounds to similar levels. Our data therefore indicate that the function of alpha3beta1 integrin as a mediator of keratinocyte migration is not essential for reepithelialization but suggest instead that alpha3beta1 integrin has a major new in vivo role as an inhibitor of Smad7 during wound healing. Moreover, our study may identify a previously undocumented function for Smad7 as a regulator of reepithelialization in vivo and implicates Smad7 as a potential novel target for the treatment of cutaneous wounds.
Asunto(s)
Epitelio/fisiología , Integrina alfa3beta1/fisiología , Proteína smad7/fisiología , Cicatrización de Heridas , Animales , Integrina alfa5beta1/fisiología , Ratones , Transducción de Señal , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.
Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Hipoxia/metabolismo , Integrina alfa3beta1/metabolismo , Neovascularización Patológica/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Western Blotting , Células Endoteliales/patología , Endotelio Vascular/patología , Femenino , Citometría de Flujo , Hipoxia/genética , Hipoxia/patología , Inmunohistoquímica , Integrina alfa3beta1/genética , Masculino , Ratones , Ratones Noqueados , Trasplante de Neoplasias , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Reacción en Cadena de la Polimerasa , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The integrin alphavbeta6 is expressed only on epithelia and then usually only during processes of tissue remodelling including cancer, where its high expression correlates with reduced survival. Thus, alphavbeta6 represents an important target for imaging and therapy of cancer and new molecular-specific targeting agents are required. We have developed A20FMDV2, a peptide derived from the VP1 coat protein of foot-and-mouth-disease virus that binds specifically and stably to alphavbeta6. Using a newly generated pair of isogenic human cell lines that differ only in alphavbeta6 expression, it was shown, using biodistribution and SPECT imaging, that indium-111-labelled A20FMDV2 locates specifically to alphavbeta6-expressing tissues in vivo, achieving at least seven-times higher retention in alphavbeta6-positive than in alphavbeta6-negative tumours. In further studies with MCF10.DCIS.COM and MCF10A.CA1a breast carcinoma cell lines, which express alphavbeta6 endogenously, the radiopeptide achieved similar levels of tumour retention and permitted excellent discriminatory imaging of tumours. Thus, A20FMDV2 can be used for molecular-specific targeting of alphavbeta6 for imaging in vivo the often more aggressive, alphavbeta6-positive cancers. In the future, A20FMDV2 could serve also to deliver therapy to these same cancers.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Animales , Femenino , Humanos , Radioisótopos de Indio/metabolismo , Radioisótopos de Indio/farmacocinética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Ácido Pentético/farmacocinética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacocinética , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodos , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
A key impediment to successful cancer therapy with adenoviral vectors is the inefficient transduction of malignant tissue in vivo. Compounding this problem is the lack of cancer-specific targets, coupled with a shortage of corresponding high-efficiency ligands, permitting selective retargeting. The epithelial cell-specific integrin alphavbeta6 represents an attractive target for directed therapy since it is generally not expressed on normal epithelium but is upregulated in numerous carcinomas, where it plays a role in tumor progression. We previously have characterized a high-affinity, alphavbeta6-selective peptide (A20FMDV2) derived from VP1 of foot-and-mouth disease virus. We generated recombinant adenovirus type 5 (Ad5) fiber knob, incorporating A20FMDV2 in the HI loop, for which we validated the selectivity of binding and functional inhibition of alphavbeta6. The corresponding alphavbeta6-retargeted virus Ad5-EGFP(A20) exhibited up to 50-fold increases in coxsackievirus- and-adenovirus-receptor-independent transduction and up to 480-fold-increased cytotoxicity on a panel of alphavbeta6-positive human carcinoma lines compared with Ad5-EGFP(WT). Using an alphavbeta6-positive (DX3-beta6) xenograft model, we observed a approximately 2-fold enhancement in tumor uptake over Ad5-EGFP(WT) following systemic delivery. Furthermore, approximately 5-fold-fewer Ad5-EGFP(A20) genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. Warfarin pretreatment, to deplete coagulation factors, did not improve tumor uptake significantly with either virus but did significantly reduce liver sequestration and hepatic toxicity. The ability of Ad5-EGFP(A20) to improve delivery to alphavbeta6, combined with its reduced hepatic tropism and toxicity, highlights its potential as a prototype virus for future clinical investigation.
Asunto(s)
Adenoviridae/genética , Antígenos de Neoplasias/metabolismo , Terapia Genética/métodos , Vectores Genéticos , Integrinas/metabolismo , Viroterapia Oncolítica , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Femenino , Genoma Viral , Humanos , Ratones , Ratones Desnudos , Unión Proteica , Receptores Virales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Pancreatic cancer is characterized by an intense stromal reaction. Reproducible three-dimensional in vitro systems for exploring interactions of the stroma with pancreatic cancer cells have not previously been available, prompting us to develop such a model. Cancer cells were grown on collagen/Matrigel and embedded with or without stromal cells (hTERT-immortalized human PS-1 stellate cells or MRC-5 fibroblasts) for 7 days. Proliferation and apoptosis, as well as important cell-cell adhesion and cytoskeleton-regulating proteins, were studied. PS-1 cells were confirmed as stellate based on the expression of key cytoskeletal proteins and lipid vesicles. Capan-1, and to a lesser extent PaCa-3, cells differentiated into luminal structures, exhibiting a central apoptotic core with a proliferating peripheral rim and an apico-basal polarity. Presence of either stromal cell type translocated Ezrin from apical (when stromal cells were absent) to basal aspects of cancer cells, where it was associated with invasive activity. Interestingly, the presence of 'normal' (not tumor-derived) stromal cells induced total tumor cell number reduction (P < 0.005) associated with a significant decrease in E-cadherin expression (P < 0.005). Conversely, beta-catenin expression was up-regulated (P < 0.01) in the presence of stromal cells with predominant cytoplasmic expression. Moreover, patient samples confirmed that these data recapitulated the clinical situation. In conclusion, pancreatic organotypic culture offers a reproducible, bio-mimetic, three-dimensional in vitro model that allows examination of the interactions between stromal elements and pancreatic cancer cells.
Asunto(s)
Cadherinas/biosíntesis , Proteínas del Citoesqueleto/biosíntesis , Neoplasias Pancreáticas/patología , beta Catenina/biosíntesis , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Técnicas de Cultivo de Órganos , Neoplasias Pancreáticas/metabolismo , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Kindler syndrome is an autosomal recessive disorder characterized by skin atrophy and blistering. It results from loss-of-function mutations in the FERMT1 gene encoding the focal adhesion protein, fermitin family homolog-1. How and why deficiency of fermitin family homolog-1 results in skin atrophy and blistering are unclear. In this study, we investigated the epidermal basement membrane and keratinocyte biology abnormalities in Kindler syndrome. We identified altered distribution of several basement membrane proteins, including types IV, VII, and XVII collagens and laminin-332 in Kindler syndrome skin. In addition, reduced immunolabeling intensity of epidermal cell markers such as beta1 and alpha6 integrins and cytokeratin 15 was noted. At the cellular level, there was loss of beta4 integrin immunolocalization and random distribution of laminin-332 in Kindler syndrome keratinocytes. Of note, active beta1 integrin was reduced but overexpression of fermitin family homolog-1 restored integrin activation and partially rescued the Kindler syndrome cellular phenotype. This study provides evidence that fermitin family homolog-1 is implicated in integrin activation and demonstrates that lack of this protein leads to pathological changes beyond focal adhesions, with disruption of several hemidesmosomal components and reduced expression of keratinocyte stem cell markers. These findings collectively provide novel data on the role of fermitin family homolog-1 in skin and further insight into the pathophysiology of Kindler syndrome.
Asunto(s)
Anomalías Múltiples/genética , Integrinas/metabolismo , Proteínas de la Membrana/genética , Mutación/genética , Proteínas de Neoplasias/genética , Anomalías Múltiples/patología , Adolescente , Adulto , Membrana Basal/metabolismo , Membrana Basal/patología , Adhesión Celular , Membrana Celular/metabolismo , Niño , Preescolar , Epidermis/metabolismo , Epidermis/patología , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Queratina-15/genética , Queratina-15/metabolismo , Queratinocitos/patología , Masculino , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Fenotipo , SíndromeRESUMEN
The demonstration that a significant proportion of patients with renal carcinomas of the clear cell type have tumour cell clumps or aggregates in venous outflow from the kidney has interest from two viewpoints. Firstly, the association of this occurrence with high VEGF-A production by the cancer seems to suggest a novel mode of 'budding' invasion where nests of tumour cells enter the dilated and mechanically fragile new vessels supplying the cancerous growth. Secondly, with the association of fragment occurrence and metastatic development, the entrance of clumps into the circulation indicates that epithelial-mesenchymal transition (EMT) is not an obligatory step for the disseminatory behaviour of all cancers.
Asunto(s)
Carcinoma de Células Renales/secundario , Neoplasias Renales/patología , Células Neoplásicas Circulantes/patología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Humanos , Neoplasias Renales/metabolismo , Proteínas de Neoplasias/biosíntesis , Células Neoplásicas Circulantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin alphavbeta6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD +1 and RGD +4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to alphavbeta6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin alphavbeta6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Proteínas de la Cápside/metabolismo , Virus de la Fiebre Aftosa/fisiología , Integrinas/metabolismo , Receptores Virales/metabolismo , Acoplamiento Viral , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
Resistance to chemotherapy is one of the principal causes of cancer mortality and is generally considered a late event in tumor progression. Although cellular models of drug resistance have been useful in identifying the molecules responsible for conferring drug resistance, most of these cellular models are derived from cell lines isolated from patients at a late stage in cancer progression. To ask at which stage in the tumorigenic progression does the cell gain the ability to acquire drug resistance, we generated a series of pre-tumorigenic and tumorigenic cells from human embryonic skin fibroblasts by introducing, sequentially, the catalytic subunit of telomerase, SV40 large T and small T oncoproteins, and an oncogenic form of ras. We show that the ability to acquire multidrug resistance (MDR) can arise before the malignant transformation stage. The minimal set of changes necessary to obtain pre-tumorigenic drug-resistant cells is expression of telomerase and inactivation of p53 and pRb. Thus, the pathways inactivated during tumorigenesis also confer the ability to acquire drug resistance. Microarray and functional studies of drug-resistant pre-tumorigenic cells indicate that the drug efflux pump P-glycoprotein is responsible for the MDR phenotype in this pre-tumorigenic cell model.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Resistencia a Antineoplásicos/fisiología , Lesiones Precancerosas/tratamiento farmacológico , Lesiones Precancerosas/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antígenos Transformadores de Poliomavirus/biosíntesis , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Transformación Celular Neoplásica , Doxorrubicina/farmacología , Embrión de Mamíferos , Fibroblastos , Expresión Génica , Humanos , Transportadores de Anión Orgánico/biosíntesis , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Lesiones Precancerosas/genética , Proteína de Retinoblastoma , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/genética , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Fenómenos Fisiológicos de la Piel/genética , Telomerasa/biosíntesis , Transfección , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Proteínas ras/biosíntesis , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Enhanced expression levels of integrin alphavbeta6 have been linked to more aggressive invasive carcinoma cell behavior and poorer clinical prognosis. However, how alphavbeta6 determines invasion and the dynamics of integrin alphavbeta6 regulation in tumor cells are poorly understood. We have identified the 35-kDa HS1-associated protein X-1 (HAX-1) protein as a novel binding partner of the beta6 cytoplasmic tail using a yeast two-hybrid screen. We show that alphavbeta6-dependent migration is blocked following small interfering RNA (siRNA)-mediated depletion of HAX-1 in oral squamous cell carcinoma cell lines. Using both siRNA and membrane-permeable peptides, we show that alphavbeta6-dependent migration and invasion require HAX-1 to bind directly to beta6 and thereby regulate clathrin-mediated endocytosis of alphavbeta6 integrins. Progression of oral cancer is associated with enhanced expression of alphavbeta6 and HAX-1 proteins in patient tissue. This report establishes that integrin endocytosis is required for alphavbeta6-dependent carcinoma cell motility and invasion and suggests that this process is an important mechanism in cancer progression.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Carcinoma de Células Escamosas/patología , Movimiento Celular/fisiología , Clatrina/metabolismo , Integrinas/metabolismo , Neoplasias de la Boca/patología , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Permeabilidad de la Membrana Celular , Regulación hacia Abajo , Endocitosis , Humanos , Integrinas/antagonistas & inhibidores , Datos de Secuencia Molecular , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Invasividad Neoplásica , Péptidos/farmacología , Unión Proteica , Proteínas/antagonistas & inhibidores , Proteínas/genética , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Worldwide oral squamous cell carcinoma (OSCC) represents about 5.5% of all malignancies, with approximately 30,000 new cases each year in the United States. The integrin alpha(v)beta(6) and the enzyme cyclooxygenase-2 (COX-2) are implicated in OSCC progression and have been suggested as possible therapeutic targets. Each protein also is reported to identify dysplasias at high risk of malignant transformation, and current clinical trials are testing the efficacy of nonsteroidal anti-inflammatory drugs (NSAID) at preventing OSCC development. Given the probable increased expression of alpha(v)beta(6) and COX-2 in OSCC and the inhibition of several integrins by NSAIDs, we investigated whether NSAIDs affected alpha(v)beta(6)-dependent cell functions. We found that expression of both alpha(v)beta(6) and COX-2 was significantly higher in OSCC compared with oral epithelial dysplasias. Neither protein preferentially identified those dysplastic lesions that became malignant. Using OSCC cell lines, modified to express varying levels of alpha(v)beta(6), we assessed the effect of COX-2 inhibition on cell invasion. We found that the COX-2 inhibitor NS398 inhibited specifically alpha(v)beta(6)-dependent, but not alpha(v)beta(6)-independent, OSCC invasion in vitro and in vivo, and this effect was modulated through prostaglandin E(2) (PGE(2))-dependent activation of Rac-1. Transient expression of constitutively active Rac-1, or addition of the COX-2 metabolite PGE(2), prevented the anti-invasive effect of NS398. Conversely, RNA interference down-regulation of Rac-1 inhibited alpha(v)beta(6)-dependent invasion. These findings suggest that COX-2 and alpha(v)beta(6) interact in promoting OSCC invasion. This is a novel mechanism that, given the ubiquity of alpha(v)beta(6) expression by head and neck cancers, raises the possibility that NSAIDs could protect against OSCC invasion.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Inhibidores de la Ciclooxigenasa 2/farmacología , Integrinas/antagonistas & inhibidores , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/patología , Animales , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/metabolismo , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Dinoprostona/farmacología , Células Epiteliales/patología , Humanos , Integrinas/biosíntesis , Integrinas/metabolismo , Ratones , Ratones Desnudos , Neoplasias de la Boca/enzimología , Invasividad Neoplásica , Nitrobencenos/farmacología , Unión Proteica , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.