RESUMEN
Anti-cancer properties of statins are controversial and possibly context dependent. Recent pathology/epidemiology studies of human lung adenocarcinoma showed reduced pro-tumourigenic macrophages associated with a shift to lower-grade tumours amongst statin users but, paradoxically, worse survival compared with that of non-users. To investigate the mechanisms involved, we have characterised mouse lung adenoma/adenocarcinoma models treated with atorvastatin. Here, we show that atorvastatin suppresses premalignant disease by inhibiting the recruitment of pro-tumourigenic macrophages to the tumour microenvironment, manifested in part by suppression of Rac-mediated CCR1 ligand secretion. However, prolonged atorvastatin treatment leads to drug resistance and progression of lung adenomas into invasive disease. Pathological progression is not driven by acquisition of additional driver mutations or immunoediting/evasion but is associated with stromal changes including the development of desmoplastic stroma containing Gr1+ myeloid cells and tertiary lymphoid structures. These findings show that any chemopreventive functions of atorvastatin in lung adenocarcinoma are overridden by stromal remodelling in the long term, thus providing mechanistic insight into the poor survival of lung adenocarcinoma patients with statin use.
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Adenocarcinoma del Pulmón , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/tratamiento farmacológico , Animales , Atorvastatina/farmacología , Atorvastatina/uso terapéutico , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Neoplasias Pulmonares/patología , Ratones , Microambiente TumoralRESUMEN
OBJECTIVE: To undertake a pilot study to develop a novel Patient-Derived-Explant (PDE) model system for use in endometrial cancer (EC) that is capable of monitoring differential drug responses in a pre-clinical setting. METHODS: Fresh tumour was obtained post-hysterectomy from 27 patients with EC. Tumours were cut into 1-3 mm3 explants that were cultured at the air-liquid interface for 16-24 h in culture media. Explants were cultured in different media conditions to optimise viability. Explants were also treated with carboplatin/paclitaxel or pembrolizumab for 24 h and processed into histology slides. Multiplexed immunofluorescence for Ki67 (proliferation marker), cPARP (apoptosis marker) and CAM 5.2 (tumour mask) was performed followed by image analysis and quantitation of biomarker expression. RESULTS: EC samples are amenable to PDE culture with preserved histological architecture and PDE viability for up to 48 h, with the addition of autologous serum in culture media facilitating EC-PDE viability. Our PDE platform provides evidence of differential drug-response to conventional chemotherapeutics and immune checkpoint inhibition, and these responses can be assessed in the context of a preserved tumour microenvironment. CONCLUSIONS: Our PDE platform represents a rapid, low-cost pre-clinical model which can be easily integrated into drug development pipelines. PDE culture preserves original tumour architecture and enables evaluation of spatial relationships in the tumour microenvironment. PDE culture has the potential for personalised drug-testing in a pre-clinical setting which is increasingly important in an era of personalised medicine in the treatment of EC.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Endometriales/terapia , Endometrio/patología , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Carboplatino/farmacología , Carboplatino/uso terapéutico , Quimioterapia Adyuvante/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Neoplasias Endometriales/genética , Neoplasias Endometriales/inmunología , Neoplasias Endometriales/patología , Endometrio/cirugía , Estudios de Factibilidad , Femenino , Heterogeneidad Genética , Humanos , Histerectomía , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Proyectos Piloto , Medicina de Precisión/métodos , Técnicas de Cultivo de Tejidos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Patient-derived explants (PDEs) represent the direct culture of fragments of freshly-resected tumour tissue under conditions that retain the original architecture of the tumour. PDEs have advantages over other preclinical cancer models as platforms for predicting patient-relevant drug responses in that they preserve the tumour microenvironment and tumour heterogeneity. At endpoint, PDEs may either be processed for generation of histological sections or homogenised and processed for 'omic' evaluation of biomarker expression. A significant advantage of spatial profiling is the ability to co-register drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. Spatial profiling of PDEs relies on the utilisation of robust immunostaining approaches for validated biomarkers and incorporation of appropriate image analysis methods to quantitatively and qualitatively monitor changes in biomarker expression in response to anti-cancer drugs. Automation of immunostaining and image analysis would provide a significant advantage for the drug discovery pipeline and therefore, here, we have sought to optimise digital pathology approaches. We compare three image analysis software platforms (QuPath, ImmunoRatio and VisioPharm) for evaluating Ki67 as a marker for proliferation, cleaved PARP (cPARP) as a marker for apoptosis and pan-cytokeratin (CK) as a marker for tumour areas and find that all three generate comparable data to the views of a histomorphometrist. We also show that Virtual Double Staining of sequential sections by immunohistochemistry results in imperfect section alignment such that CK-stained tumour areas are over-estimated. Finally, we demonstrate that multi-immunofluorescence combined with digital image analysis is a superior method for monitoring multiple biomarkers simultaneously in tumour and stromal areas in PDEs.
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Antineoplásicos/uso terapéutico , Monitoreo de Drogas/métodos , Interpretación de Imagen Asistida por Computador/métodos , Inmunohistoquímica/métodos , Neoplasias , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Biología Computacional , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
The use of a low aerosol dispersion ablation chamber within a laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS) setup allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle-induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS), can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.
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Cisplatino/uso terapéutico , Neoplasias Pulmonares/química , Neoplasias Pulmonares/tratamiento farmacológico , Espectrometría de Masas/métodos , Antineoplásicos/uso terapéutico , Carbono/química , Carcinoma de Pulmón de Células no Pequeñas , Humanos , Terapia por Láser , Esferoides Celulares , Técnicas de Cultivo de TejidosRESUMEN
Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adjuncts to provide more precise prognostic information. Simple, low-cost biomarker assays, such as those based on immunohistochemistry, have strong translational potential. 5-hydroxymethylcytosine (5 hmC) shows prognostic potential in melanoma but prior studies were small. We, therefore, analysed 5 hmC in a retrospective cohort to provide external validation of its prognostic value. Two hundred primary melanomas were evaluated for 5 hmC expression using immunohistochemistry. The primary objective was to assess the effect on overall survival while controlling for important confounders. Univariable and multivariable analyses were performed. REMARK guidelines were followed. The 5 hmC immunohistochemistry scoring showed very strong inter-observer agreement (ICC 0.88) and expression was significantly related to age, site, Breslow thickness, ulceration, mitotic rate, and stage. Kaplan-Meier analysis showed 5 hmC was associated with metastasis-free, melanoma-specific, and overall survival, P<0.0001 for each. In univariable Cox proportional hazards models, 5 hmC hazard ratios were significant and remained so in a multivariable model. A two-step cox model was created using stage and 5 hmC, as stage is the gold standard for clinical practice. The addition of 5 hmC produced significant improvement in the model and 5 hmC and stage were independent significant predictors. This is the largest study of the prognostic value of 5 hmC immunohistochemistry in melanoma. The 5 hmC scoring was easily and reproducibly performed and it was an independent predictor of metastasis-free survival, melanoma-specific survival, and overall survival. This work supports further development of 5 hmC as a prognostic biomarker and suggests that it could add more precision to American Joint Committee on Cancer staging.
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5-Metilcitosina/análogos & derivados , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Piel/metabolismo , 5-Metilcitosina/metabolismo , Factores de Edad , Anciano , Femenino , Humanos , Masculino , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Piel/patología , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Tasa de SupervivenciaRESUMEN
Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n=79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P=0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (P<0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P=0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P=0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.
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Biomarcadores de Tumor/genética , Melanoma/genética , MicroARNs/genética , Neoplasias Cutáneas/genética , Anciano , Femenino , Estudios de Asociación Genética , Humanos , Modelos Logísticos , Masculino , Melanoma/secundario , Persona de Mediana Edad , Análisis Multivariante , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND: Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a "liquid biopsy." METHODS: We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α-positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). RESULTS: Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. CONCLUSIONS: Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease.
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Neoplasias de la Mama/genética , Análisis Mutacional de ADN/métodos , Receptor alfa de Estrógeno/genética , Mutación , Células Neoplásicas Circulantes , Neoplasias de la Mama/patología , Fosfatidilinositol 3-Quinasa Clase I , Receptor alfa de Estrógeno/sangre , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Células Neoplásicas Circulantes/patología , Fosfatidilinositol 3-Quinasas/genética , Reproducibilidad de los Resultados , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp(®) DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.
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Recolección de Muestras de Sangre/métodos , Neoplasias de la Mama/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/aislamiento & purificación , MicroARNs/genética , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , ADN de Neoplasias/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Juego de Reactivos para Diagnóstico , Reproducibilidad de los ResultadosRESUMEN
Aberrant expression of embryonic epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) in epithelial cells triggers EMT, neoplastic transformation, stemness, and metastatic dissemination. We found that regulation and functions of EMT-TFs are different in malignant melanoma. SNAIL2 and ZEB2 transcription factors are expressed in normal melanocytes and behave as tumor-suppressor proteins by activating an MITF-dependent melanocyte differentiation program. In response to NRAS/BRAF activation, EMT-TF network undergoes a profound reorganization in favor of TWIST1 and ZEB1. This reversible switch cooperates with BRAF in promoting dedifferentiation and neoplastic transformation of melanocytes. We detected EMT-TF reprogramming in late-stage melanoma in association with enhanced phospho-ERK levels. This switch results in E-cadherin loss, enhanced invasion, and constitutes an independent factor of poor prognosis in melanoma patients.
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Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , Animales , Antígenos CD , Cadherinas/metabolismo , Diferenciación Celular , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Proteínas de Homeodominio/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Melanocitos/citología , Ratones , Ratones Desnudos , Proteínas Nucleares/metabolismo , Fosforilación , Pronóstico , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
A minority of melanocytic lesions cannot confidently be classified as benign or malignant on histopathological examination, causing diagnostic uncertainty. DNA copy number changes can be used to distinguish nevi from melanoma, although the use of FFPE tissue can pose technical challenges. DNA copy number assays, called duplex ratio tests, have been developed with duplex real-time PCR, using a simple method with potential for high throughput. Five duplex ratio test assays targeting loci with common DNA copy number changes in melanoma were designed and tested using DNA extracted from FFPE samples microdissected from melanoma, common nevi, benign tonsil (10 each), and two melanoma cell lines. The assays proved accurate when DNA extracted from fresh and FFPE melanoma cell lines were compared (intraclass correlation coefficient, 0.99) and gave precise results when repeated on DNA from FFPE tissue (intraclass correlation coefficient range, 0.90 to 0.96). In combination, duplex ratio test values from three of the assays distinguished between the nevi and melanomas with 100% sensitivity (95% CI, 69.1% to 100%) and 100% specificity (95% CI, 69.1% to 100%). Duplex ratio test assays have been shown to be accurate and precise and can distinguish melanomas from common nevi using DNA from FFPE tissue. Appropriately designed assays could have value in assessment of other cancers.
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Variaciones en el Número de Copia de ADN , Melanoma/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Sitios Genéticos , Humanos , Masculino , Melanoma/genética , Persona de Mediana Edad , Nevo/diagnóstico , Nevo/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenAsunto(s)
Biomarcadores de Tumor/sangre , Melanoma/patología , MicroARNs/sangre , Neoplasias Cutáneas/patología , Carga Tumoral , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Masculino , Melanocitos/patología , Melanoma/sangre , Persona de Mediana Edad , Nevo/sangre , Nevo/patología , Sensibilidad y Especificidad , Neoplasias Cutáneas/sangre , Adulto JovenRESUMEN
Prognostic tissue markers in melanoma Prognosis for patients diagnosed with cutaneous melanoma is currently based upon histopathological features alone, although tumours which are morphologically similar can behave differently. Numerous putative biomarkers have been identified in an attempt to aid prognostication for primary melanoma, using methods which include immunhistochemistry, polymerase chain reaction (PCR), array comparative genomic hybridization (CGH) and gene expression arrays. Despite this wide body of research, no biomarkers for prognosis in melanoma have been translated or are close to translation into clinical practice. In this review selected prognostic biomarkers are evaluated and the factors influencing successful biomarker translation, including phases of biomarker development and study design, are explored in an attempt to highlight the current gap between prognostic melanoma biomarker research and clinical translation.
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Biomarcadores de Tumor/metabolismo , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Hibridación Genómica Comparativa , Perfilación de la Expresión Génica , Marcadores Genéticos , Humanos , Inmunohistoquímica/métodos , Melanoma/genética , Reacción en Cadena de la Polimerasa , Pronóstico , Neoplasias Cutáneas/genéticaRESUMEN
Tenascin-C (TNC) is an extracellular matrix glycoprotein which is frequently up-regulated in a variety of pathological conditions including chronic inflammation and cancer. TNC has been implicated in the modulation of cell migration, proliferation, invasion and angiogenesis. Multiple isoforms of TNC can be generated through the alternative splicing of nine exons located in the fibronectin type III region of the molecule. The profile of isoforms expressed differs between cancers and normal breast, with the fully truncated TNC isoform being predominant in normal and benign tissues and higher molecular weight isoforms induced predominantly in cancer. The addition of extra domains within the fibronectin type III repeat domain greatly affects TNC function with multiple exon combinations available for splicing. Exons 14 and 16 are considered to be tumour-associated and have been shown to affect breast cell line invasion and growth in vitro to a greater extent than the full-length TNC isoform. This mini review will provide a summary of the literature to date regarding the expression of TNC isoforms in the breast and also discuss more recent developments in the field regarding exon AD1.
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Neoplasias de la Mama/metabolismo , Mama/metabolismo , Tenascina/biosíntesis , Animales , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Metástasis de la Neoplasia , Isoformas de Proteínas , Tenascina/genéticaRESUMEN
INTRODUCTION: Tenascin-C (TNC) is a large extracellular matrix glycoprotein that shows prominent stromal expression in many solid tumours. The profile of isoforms expressed differs between cancers and normal breast, with the two additional domains AD1 and AD2 considered to be tumour associated. The aim of the present study was to investigate expression of AD1 and AD2 in normal, benign and malignant breast tissue to determine their relationship with tumour characteristics and to perform in vitro functional assays to investigate the role of AD1 in tumour cell invasion and growth. METHODS: Expression of AD1 and AD2 was related to hypoxanthine phosphoribosyltransferase 1 as a housekeeping gene in breast tissue using quantitative RT-PCR, and the results were related to clinicopathological features of the tumours. Constructs overexpressing an AD1-containing isoform (TNC-14/AD1/16) were transiently transfected into breast carcinoma cell lines (MCF-7, T-47 D, ZR-75-1, MDA-MB-231 and GI-101) to assess the effect in vitro on invasion and growth. Statistical analysis was performed using a nonparametric Mann-Whitney test for comparison of clinicopathological features with levels of TNC expression and using Jonckheere-Terpstra trend analysis for association of expression with tumour grade. RESULTS: Quantitative RT-PCR detected AD1 and AD2 mRNA expression in 34.9% and 23.1% of 134 invasive breast carcinomas, respectively. AD1 mRNA was localised by in situ hybridisation to tumour epithelial cells, and more predominantly to myoepithelium around associated normal breast ducts. Although not tumour specific, AD1 and AD2 expression was significantly more frequent in carcinomas in younger women (age ≤40 years; P < 0.001) and AD1 expression was also associated with oestrogen receptor-negative and grade 3 tumours (P < 0.05). AD1 was found to be incorporated into a tumour-specific isoform, not detected in normal tissues. Overexpression of the TNC-14/AD1/16 isoform significantly enhanced tumour cell invasion (P < 0.01) and growth (P < 0.01) over base levels. CONCLUSIONS: Together these data suggest a highly significant association between AD-containing TNC isoforms and breast cancers in younger women (age ≤40 years), which may have important functional significance in vivo.
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Neoplasias de la Mama/genética , Proliferación Celular , Perfilación de la Expresión Génica , Tenascina/genética , Adulto , Factores de Edad , Sitios de Unión/genética , Western Blotting , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Epitelio/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Invasividad Neoplásica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tenascina/metabolismoRESUMEN
INTRODUCTION: The stromal microenvironment has a profound influence on tumour cell behaviour. In tumours, the extracellular matrix (ECM) composition differs from normal tissue and allows novel interactions to influence tumour cell function. The ECM protein tenascin-C (TNC) is frequently up-regulated in breast cancer and we have previously identified two novel isoforms - one containing exon 16 (TNC-16) and one containing exons 14 plus 16 (TNC-14/16). METHODS: The present study has analysed the functional significance of this altered TNC isoform profile in breast cancer. TNC-16 and TNC-14/16 splice variants were generated using PCR-ligation and over-expressed in breast cancer cells (MCF-7, T47D, MDA-MD-231, MDA-MB-468, GI101) and human fibroblasts. The effects of these variants on tumour cell invasion and proliferation were measured and compared with the effects of the large (TNC-L) and fully spliced small (TNC-S) isoforms. RESULTS: TNC-16 and TNC-14/16 significantly enhanced tumour cell proliferation (P < 0.05) and invasion, both directly (P < 0.01) and as a response to transfected fibroblast expression (P < 0.05) with this effect being dependent on tumour cell interaction with TNC, because TNC-blocking antibodies abrogated these responses. An analysis of 19 matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases 1 to 4 (TIMP 1 to 4) revealed that TNC up-regulated expression of MMP-13 and TIMP-3 two to four fold relative to vector, and invasion was reduced in the presence of MMP inhibitor GM6001. However, this effect was not isoform-specific but was elicited equally by all TNC isoforms. CONCLUSIONS: These results demonstrate a dual requirement for TNC and MMP in enhancing breast cancer cell invasion, and identify a significant role for the tumour-associated TNC-16 and TNC-14/16 in promoting tumour invasion, although these isoform-specific effects appear to be mediated through MMP-independent mechanisms.
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Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Metaloproteinasas de la Matriz/metabolismo , Tenascina/fisiología , Empalme Alternativo , Western Blotting , Neoplasias de la Mama/genética , Adhesión Celular , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Metaloproteinasas de la Matriz/genética , Invasividad Neoplásica , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Regulación hacia Arriba , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
PURPOSE: Wnt ligands play a major role in development and are important in cancer. Expression microarray analysis correlates one member of this family, WNT5A, to a subclass of melanomas with increased motility and invasion. There are no large studies of clinical samples primarily addressing the importance of WNT5A in melanoma progression or outcome. Therefore, this study aimed to assess the protein expression of WNT5A during melanoma progression and its effect on outcome. EXPERIMENTAL DESIGN: Expression of WNT5A was determined in a series of 59 primary melanomas with matched metastases. To provide a benchmark of progression against which to assess WNT5A, expression of p16(ink4a) was analyzed, as this has been previously well documented in melanoma. The effect of WNT5A protein expression on outcome was assessed in 102 melanomas. RESULTS: Cytoplasmic WNT5A showed a trend of increasing expression with melanoma progression (P = 0.013), whereas there was diminishing p16(ink4a) expression (P = 0.006). Nevi showed relatively strong WNT5A expression. Strong cytoplasmic WNT5A was an independent risk factor for reduced metastasis-free and overall survival in multivariate analysis (P = 0.001 and 0.003, respectively). CONCLUSION: Cytoplasmic WNT5A increases with melanoma progression and strong expression is associated with poor outcome.
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Melanoma/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas Wnt/metabolismo , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Citoplasma/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Neoplasias Cutáneas/patología , Análisis de Supervivencia , Proteína Wnt-5aRESUMEN
Fos proteins have been implicated in control of tumorigenesis-related genetic programs including invasion, angiogenesis, cell proliferation and apoptosis. In this study, we demonstrate that c-Fos is able to induce mesenchymal transition in murine tumorigenic epithelial cell lines. Expression of c-Fos in MT1TC1 cells led to prominent alterations in cell morphology, increased expression of mesenchymal markers, vimentin and S100A4, DNA methylation-dependent down-regulation of E-cadherin and abrogation of cell-cell adhesion. In addition, c-Fos induced a strong beta-catenin-independent proliferative response in MT1TC1 cells and stimulated cell motility, invasion and adhesion to different extracellular matrix proteins. To explore whether loss of E-cadherin plays a role in c-Fos-mediated mesenchymal transition, we expressed wild-type E-cadherin and two different E-cadherin mutants in MT1TC1/c-fos cells. Expression of wild-type E-cadherin restored epithelioid morphology and enhanced cellular levels of catenins. However, exogenous E-cadherin did not influence expression of c-Fos-dependent genes, only partly suppressed growth of MT1TC1/c-fos cells and produced no effect on c-Fos-stimulated cell motility and invasion in matrigel. On the other hand, re-expression of E-cadherin specifically negated c-Fos-induced adhesion to collagen type I, but not to laminin or fibronectin. Of interest, mutant E-cadherin which lacks the ability to form functional adhesive complexes had an opposite, potentiating effect on cell adhesion to collagen I. These data suggest that cell adhesion to collagen I is regulated by the functional state of E-cadherin. Overall, our data demonstrate that, with the exception of adhesion to collagen I, c-Fos is dominant over E-cadherin in relation to the aspects of mesenchymal transition assayed in this study.
Asunto(s)
Adenocarcinoma/patología , Cadherinas/genética , Modelos Biológicos , Invasividad Neoplásica/genética , Proteínas Proto-Oncogénicas c-fos/fisiología , Adenocarcinoma/genética , Animales , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Metilación de ADN , Epigénesis Genética , Células Epiteliales/patología , Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Mesodermo/patología , Ratones , Mutación , Invasividad Neoplásica/patología , Regiones Promotoras Genéticas , beta Catenina/metabolismoRESUMEN
PURPOSE: BRAF mutations are present in two thirds of cutaneous melanomas and many of the rest have NRAS mutations. However, cutaneous melanoma is a heterogeneous disease with many clinicopathologic subtypes. Of these, the majority fits into four categories: superficial spreading, nodular, lentigo maligna, and acral lentiginous melanoma (ALM). Thus far, there is very limited data combining BRAF and NRAS mutation analysis to explore differences between cutaneous melanoma subtypes. The aim of this study was to address this issue. EXPERIMENTAL DESIGN: The frequency of BRAF and NRAS hotspot mutations, in exons 15 and 2, respectively, was assessed in 59 cutaneous melanomas comprising superficial spreading, nodular, lentigo maligna, and ALM using single-strand conformational polymorphism and RFLP-PCR analysis. RESULTS: Only 2 of 21 (9.5%) ALM showed BRAF exon 15 mutation compared with 9 of 14 (64.3%) superficial spreading malignant melanomas, 4 of 11 (36.4%) nodular melanomas, and 7 of 13 (53.4%) lentigo maligna melanomas (P < 0.01). However, our key finding is that the combined analysis of BRAF exon 15 and NRAS exon 2 showed that there were no significant differences in the overall mutation frequency between subtypes. In particular, 9 of 19 (47.4%) ALM without BRAF exon 15 mutation had an NRAS exon 2 mutation. CONCLUSIONS: We show that the overall BRAF/NRAS frequency in mutation hotspots is not significantly different among cutaneous melanoma subtypes. These data show that mitogen-activated protein kinase pathway activation may be important in all major subtypes of cutaneous melanoma, although the mechanism by which this is achieved varies.
Asunto(s)
Genes ras/genética , Melanoma/clasificación , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/clasificación , Neoplasias Cutáneas/genética , Adulto , Anciano , Anciano de 80 o más Años , Exones , Femenino , Frecuencia de los Genes , Humanos , Masculino , Melanoma/diagnóstico , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa/métodos , Neoplasias Cutáneas/diagnósticoRESUMEN
Cutaneous melanoma (CM) is the most lethal form of skin cancer. Along with some benign melanocytic tumors, the majority shows BRAF or NRAS mutation, but it is not known whether these are essential to all forms of melanocytic neoplasia. We screened 79 melanocytic tumors of different types for BRAF and NRAS mutations and looked at MAPK pathway activity using immunohistochemistry in a subset. Significant differences in BRAF exon 15 mutation frequency were found: 14/16 (87.5%) in common acquired naevi (CANs), 9/12 (75%) in CMs, 0/26 in Spitz naevi and 3/25 (12%) in blue naevi (p < 0.01). We looked at whether Spitz and blue naevi showed a compensatory increase in BRAF exon 11 and/or NRAS exons 1 and 2 mutations to account for the low BRAF exon 15 mutation frequency. NRAS mutations were found in only 1/16 (6.3%) Spitz naevi and 0/15 blue naevi. In addition, NRAS mutations were found in 2/11 (18.2%) CANs and 3/12 (25%) CMs. None of the tumors showed BRAF exon 11 mutations. Despite their low combined BRAF and NRAS mutation frequency, Spitz naevi showed strong MAPK pathway activation as measured by cytoplasmic expression of dually phosphorylated ERK1/2, while blue naevi had weak pathway activation. We conclude that BRAF and NRAS mutations are not necessary for melanocytic tumor development and that some types of tumor must arise by alternative mechanisms.