RESUMEN
Cancer/testis (CT) genes are predominantly expressed in human germ line cells, but not somatic tissues, and frequently become activated in different cancer types. Several CT antigens have already proved to be useful biomarkers and are promising targets for therapeutic cancer vaccines. The aim of the present study was to investigate the expression of CT antigens in breast cancer. Using previously generated massively parallel signature sequencing (MPSS) data, together with 9 publicly available gene expression datasets, the expression pattern of CT antigens located on the X chromosome (CT-X) was interrogated. Whereas a minority of unselected breast cancers was found to contain CT-X transcripts, a significantly higher expression frequency was detected in estrogen and progesterone receptor (ER) negative breast cancer cell lines and primary breast carcinomas. A coordinated pattern of CT-X antigen expression was observed, with MAGEA and NY-ESO-1/CTAG1B being the most prevalent antigens. Immunohistochemical staining confirmed the correlation of CT-X antigen expression and ER negativity in breast tumors and demonstrated a trend for their coexpression with basal cell markers. Because of the limited therapeutic options for ER-negative breast cancers, vaccines based on CT-X antigens might prove to be useful.
Asunto(s)
Antígenos de Neoplasias/genética , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Etiquetas de Secuencia Expresada , Femenino , Humanos , Inmunohistoquímica , Proteínas de la Membrana/genética , Metástasis de la Neoplasia/patología , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Análisis de Matrices TisularesRESUMEN
Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteoglicanos de Heparán Sulfato/metabolismo , Melanoma/patología , Proteínas Proto-Oncogénicas/química , Neoplasias Cutáneas/patología , Proteínas Wnt/química , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteoglicanos de Heparán Sulfato/química , Humanos , Melanoma/metabolismo , Metástasis de la Neoplasia , Proteínas Recombinantes/química , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Sindecano-1/metabolismo , Sindecano-4/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a , Cicatrización de HeridasRESUMEN
Ultraviolet (UV) irradiation plays a pivotal role in human skin carcinongenesis. Preclinically, systemically and topically applied green tea extract (GTE) has shown reduction of UV-induced (i) erythema, (ii) DNA damage, (iii) formation of radical oxygen species and (iv) downregulation of numerous factors related to apoptosis, inflammation, differentiation and carcinogenesis. In humans, topical GTE has so far only been tested in limited studies, with usually very high GTE concentrations and over short periods of time. Both chemical stability of GTE and staining properties of highly concentrated green tea polyphenols limit the usability of highly concentrated green tea extracts in cosmetic products. The present study tested the utility of stabilized low-dose GTE as photochemopreventive agents under everyday conditions. We irradiated with up to 100 mJ/cm(2) of UVB light skin patches which were pretreated with either OM24-containing lotion or a placebo lotion. Biopsies were taken from both irradiated and un-irradiated skin for both immunohistochemistry and DNA microarray analysis. We found that while OM24 treatment did not significantly affect UV-induced erythema and thymidine dimer formation, OM24 treatment significantly reduced UV-induced p53 expression in keratinocytes. We also found that OM24 treatment significantly reduced the number of apoptotic keratinocytes (sunburn cells and TUNEL-positive cells). Carefully controlled DNA microarray analyses showed that OM24 treatment does not induce off-target changes in gene expression, reducing the likelihood of unwanted side-effects. Topical GTE (OM24) reduces UVB-mediated epithelial damage already at low, cosmetically usable concentrations, without tachyphylaxis over 5 weeks, suggesting GTE as suitable everyday photochemopreventive agents.
Asunto(s)
Apoptosis , Piel/metabolismo , Té , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta , Adulto , Anciano , Biopsia , Daño del ADN , Femenino , Humanos , Queratinocitos/metabolismo , Masculino , Persona de Mediana Edad , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
BACKGROUND: The C-terminal binding protein 1 (CtBP1) is a known co-repressor of gene transcription. We recently revealed that CtBP1 expression is lost in melanoma cells and melanoma inhibitory activity (MIA) expression is subsequently increased. The present study was performed to evaluate a more general role of CtBP1 in human melanoma and identify further CtBP1-regulated target genes. METHODS: Sequence analysis and expression profile of CtBP1 in melanoma cell lines were done by PCR. Boyden Chamber assays and co-immunoprecipitation were performed to investigate the functional role of CtBP1. Gene expression analysis and micro array data were used to define target genes. RESULTS: Interestingly, we detected an alternative splice product of CtBP1 with unknown function whose expression is induced at reduction of full length CtBP1. Overexpression of full length CtBP1 in melanoma cells had no effect on cell proliferation but did influence cell migration and invasiveness. To understand the effect of CtBP1 we identified putative LEF/TCF target genes found to be strongly expressed in melanoma using DNA microarray analysis. We focused on fourteen genes not previously associated with melanoma. Detailed analysis revealed that most of these were known to be involved in tumor metastasis. Eleven genes had expression profiles associated with melanoma cell invasiveness. CONCLUSION: In summary, this study revealed that reduction of CtBP1 expression is correlated with migratory, invasive potential of melanoma cells.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Melanoma/patología , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Secuencia de Bases , Línea Celular Tumoral , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Melanoma/metabolismo , Datos de Secuencia Molecular , Invasividad NeoplásicaRESUMEN
Cutaneous melanoma is the most aggressive of cutaneous neoplasms. Identifying patients with an increased risk for the development of metastases is critical. This study investigates phospho-Smad2, a central factor of the transforming growth factor beta pathway, on formalin-fixed, paraffin-embedded tissues from 60 primary cutaneous melanomas (Breslow >1 mm), for its candidacy for being a prognostic marker in primary cutaneous melanoma. Phospho-Smad2 positivity was assessed for correlation with clinical parameters including Breslow index, melanoma type, survival, development of metastases, sentinel lymph node status and age. Phospho-Smad2 positivity was not associated with survival or development of metastases, suggesting that it would not be a useful prognostic marker. Despite this, we found phospho-Smad2 positivity to be correlated with low tumour thickness, indicating that as the primary tumour grows there is an increased inhibition of transforming growth factor beta signalling resulting in suppressed Smad2 phosphorylation. Additionally, phosphorylation of Smad2 in neighbouring melanoma cells and keratinocytes was interrelated, which is a further indication that Smad2 phosphorylation in primary melanoma is affected by local area microenvironmental factors. We hypothesize that the observed decrease in transforming growth factor beta signalling in thicker primary melanomas is due to the increased production of signalling inhibitors.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Melanoma/diagnóstico , Melanoma/metabolismo , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/metabolismo , Proteína Smad2/biosíntesis , Adulto , Factores de Edad , Anciano , Femenino , Formaldehído/farmacología , Humanos , Inmunofenotipificación , Masculino , Melanoma/inmunología , Persona de Mediana Edad , Metástasis de la Neoplasia , Parafina/farmacología , Neoplasias Cutáneas/inmunologíaRESUMEN
We have previously reported a model for melanoma progression in which oscillation between melanoma cell phenotypes characterized by invasion or proliferation is fundamental to tumor heterogeneity and disease progression. In this study we examine the possible role of hypoxia as one of the microenvironmental influences driving metastatic progression by promoting a switch from a proliferative to an invasive phenotype. Immunohistochemistry on primary human cutaneous melanoma biopsies showed intratumoral heterogeneity for cells expressing melanocytic markers, and a loss of these markers correlated with hypoxic regions. Furthermore, we show that the downregulation of melanocytic markers is dependent on hypoxia inducible factor 1α (HIF1α), a known regulator of the hypoxic response. In vitro invasion assays showed that a hypoxic environment increases the invasiveness of proliferative melanoma cell cultures in a HIF1α-dependent manner. In contrast, invasive phenotype melanoma cells showed no increase in invasive potential upon exposure to hypoxia. Thus, exposure of proliferative melanoma cells to hypoxic microenvironments is sufficient, in a HIF1α-dependent manner, to downregulate melanocytic marker expression and increase their invasive potential.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/patología , Melanoma/patología , Neoplasias Cutáneas/patología , Microambiente Tumoral/fisiología , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Humanos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Antígeno MART-1/genética , Antígeno MART-1/metabolismo , Melanoma/genética , Melanoma/metabolismo , Invasividad Neoplásica , Fenotipo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Células Tumorales CultivadasRESUMEN
The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells.
Asunto(s)
Cadherinas/metabolismo , Caveolina 1/metabolismo , Melanoma/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/secundario , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Metástasis de la Neoplasia , Neuropéptidos/metabolismo , Fenotipo , Neoplasias Cutáneas/metabolismo , beta Catenina/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Melanoma is one of the most aggressive types of human cancers, and the mechanisms underlying melanoma invasive phenotype are not completely understood. Here, we report that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in de novo biosynthesis of purine nucleotides, was downregulated in the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR downregulates the amounts of several GTP-bound (active) Rho-GTPases and suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade in vitro, and grow as tumor xenografts in vivo. Mechanistically, we demonstrated that GMPR partially depletes intracellular GTP pools. Pharmacological inhibition of de novo GTP biosynthesis suppressed whereas addition of exogenous guanosine increased invasion of melanoma cells as well as cells from other cancer types. Our data identify GMPR as a melanoma invasion suppressor and establish a link between guanosine metabolism and Rho-GTPase-dependent melanoma cell invasion.
Asunto(s)
GMP-Reductasa/metabolismo , Melanoma/enzimología , Nucleósidos de Purina/biosíntesis , Animales , Línea Celular Tumoral , Movimiento Celular , Matriz Extracelular/metabolismo , GMP-Reductasa/antagonistas & inhibidores , GMP-Reductasa/genética , Guanosina Trifosfato/metabolismo , Células HCT116 , Humanos , IMP Deshidrogenasa/metabolismo , Melanoma/metabolismo , Melanoma/patología , Ratones , Fenotipo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Trasplante Heterólogo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.
Asunto(s)
Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Perfilación de la Expresión Génica , Melanoma/clasificación , Neoplasias Cutáneas/clasificación , Proliferación Celular , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias/genética , Estudios de Asociación Genética , Humanos , Melanoma/genética , Melanoma/patología , Análisis por Micromatrices , Invasividad Neoplásica , Fenotipo , Reproducibilidad de los Resultados , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Distribución Tisular , Células Tumorales Cultivadas/clasificaciónRESUMEN
Recent observations suggest that melanoma cells drive disease progression by switching back and forth between phenotypic states of proliferation and invasion. Phenotype switching has been linked to changes in Wnt signalling, and we therefore looked for cell phenotype-specific differences in the levels and activity of ß-catenin and its LEF/TCF co-factors. We found that while cytosolic ß-catenin distribution is phenotype-specific (membrane-associated in proliferative cells and cytosolic in invasive cells), its nuclear distribution and activity is not. Instead, the expression patterns of two ß-catenin co-factors, LEF1 and TCF4, are both phenotype-specific and inversely correlated. LEF1 is preferentially expressed by differentiated/proliferative phenotype cells and TCF4 by dedifferentiated/invasive phenotype cells. Knock-down experiments confirmed that these co-factors are important for the phenotype-specific expression of M-MITF, WNT5A and other genes and that LEF1 suppresses TCF4 expression independently of ß-catenin. Our data show that melanoma cell phenotype switching behaviour is regulated by differential LEF1/TCF4 activity.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factor de Unión 1 al Potenciador Linfoide/genética , Melanoma/genética , Melanoma/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Modelos Biológicos , Invasividad Neoplásica , Fenotipo , Factor de Transcripción 4 , Factores de Transcripción/metabolismo , Proteínas Wnt/farmacología , beta Catenina/metabolismoRESUMEN
We recently identified GLI2, the most active of GLI transcription factors, as a direct TGF-ß/SMAD target, whose expression in melanoma cells is associated with increased invasiveness and metastatic capacity. In this work, we provide evidence that high GLI2 expression is inversely correlated with that of the melanocyte-specific transcription factor M-microphthalmia transcription factor (M-MITF) and associated transcriptional program. GLI2-expressing cell lines were characterized by the loss of M-MITF-dependent melanocytic differentiation markers and reduced pigmentation. The balance between M-MITF and GLI2 expression did not correlate with the presence or absence of BRAF-activating mutations, but rather was controlled by two distinct pathways: the TGF-ß pathway, which favors GLI2 expression, and the protein kinase A (PKA)/cAMP pathway, which pushes the balance toward high M-MITF expression. Furthermore, overexpression and knockdown experiments demonstrated that GLI2 and M-MITF reciprocally repress each other's expression and control melanoma cell invasion in an opposite manner. These findings thus identify GLI2 as a critical transcription factor antagonizing M-MITF function to promote melanoma cell phenotypic plasticity and invasive behavior.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Melanoma/genética , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Melanoma/fisiopatología , Ratones , Ratones Desnudos , Factor de Transcripción Asociado a Microftalmía/genética , Invasividad Neoplásica , Proteínas Nucleares/genética , Pigmentación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Neoplasias Cutáneas/fisiopatología , Proteínas Smad/metabolismo , Tejido Subcutáneo/efectos de los fármacos , Tejido Subcutáneo/patología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Gli2 con Dedos de ZincRESUMEN
Oncogenic mutations within the MAPK pathway are frequent in melanoma, and targeting of MAPK signaling has yielded spectacular responses in a significant number of patients that last for several months before relapsing. We investigated the effects of two different inhibitors of MAPK signaling in proliferative and invasive melanoma cell cultures with various mutations in the MAPK pathway. Proliferative melanoma cells were more susceptible to pathway inhibition than invasive phenotype cells, irrespective of BRAF mutation status, while invasive phenotype cell response was dependent on BRAF mutation status. Critically, MAPK pathway inhibition of proliferative phenotype cells resulted in acquisition of invasive phenotype characteristics. These results show that melanoma cell phenotype is an important factor in MAPK pathway inhibition response. This suggests that while current therapeutic strategies target proliferative melanoma cells, future approaches should also account for the invasive phenotype population.
Asunto(s)
Melanoma/genética , Melanoma/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Fenotipo , Proteínas Proto-Oncogénicas B-raf/genética , Quinasas raf/antagonistas & inhibidores , Adulto , Anciano , Proliferación Celular , Inhibidores Enzimáticos/uso terapéutico , Femenino , Perfilación de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Análisis por Micromatrices , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Invasividad Neoplásica , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
Coverage on: Berger, M.F. et al. (2010).Integrative analysis of the melanoma transcriptome.Genome Res. (doi: 10.1101/gr.103697.109).
Asunto(s)
Melanoma/genética , Análisis de Secuencia de ADN/métodos , Proteínas de Fusión bcr-abl/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico/genética , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Tumours comprise multiple phenotypically distinct subpopulations of cells, some of which are proposed to possess stem cell-like properties, being able to self-renew, seed and maintain tumours, and provide a reservoir of therapeutically resistant cells. Here, we use melanoma as a model to explore the validity of the cancer stem cell hypothesis in the light of accumulating evidence that melanoma progression may instead be driven by phenotype-switching triggered by genetic lesions that impose an increased sensitivity to changes in the tumour microenvironment. Although at any given moment cells within a tumour may exhibit differentiated, proliferative or invasive phenotypes, an ability to switch phenotypes implies that most cells will have the potential to adopt a stem cell-like identity. Insights into the molecular events underpinning phenotype-switching in melanoma highlight the close relationship between signalling pathways that generate, maintain and activate melanocyte stem cells as well as the inverse correlation between proliferation and invasive potentials. An understanding of phenotype-switching in melanoma, and in particular the signalling events that regulate the expression of the microphthalmia-associated transcription factor Mitf, points to new therapeutic opportunities aimed at eradicating therapeutically resistant stem cell-like melanoma cells.
Asunto(s)
Melanoma/patología , Células Madre Neoplásicas/patología , Animales , Humanos , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/genética , Melanoma/terapia , Factor de Transcripción Asociado a Microftalmía/metabolismo , Células Madre Neoplásicas/metabolismo , Fenotipo , Lesiones Precancerosas/patologíaRESUMEN
To date there is no effective therapy for metastatic melanoma and at the molecular level the disease progression is poorly understood. A recent study by our group led to the development of a novel phenotype switching model for melanoma progression, wherein cells transition back-and-forth between states of proliferation and invasion to drive disease progression. To explore the model's clinical relevance we interrogated phenotype-specific expression patterns in human melanoma patient material. A matched primary/metastasis pair from a human melanoma patient was obtained and immunohistochemically stained for proliferative and invasive phenotype markers. These were also stained for hypoxia and blood vessel markers. Proliferative phenotype markers Melan-A and Mitf showed consistent anti-correlation with invasive phenotype marker Wnt5A and hypoxia marker Glut-1. These also correlated with observed intra-tumoural vascularization patterns. Similar pattern distributions were present in both primary and metastasis samples. Strikingly, we observed that late phase metastatic melanoma cells adopt morphologies and behaviours identical to very early phase cells. The expression patterns observed closely matched expectations derived from previous in vitro and xenografting experiments. These results highlight the likelihood that disease progression involves melanoma cells retaining the capacity to regulate the expression of metastatic potential critical factors according to changing microenvironmental conditions.
Asunto(s)
Melanoma/patología , Neoplasias Cutáneas/patología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Progresión de la Enfermedad , Femenino , Neoplasias de la Vesícula Biliar/genética , Neoplasias de la Vesícula Biliar/metabolismo , Neoplasias de la Vesícula Biliar/patología , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Antígeno MART-1 , Melanoma/genética , Melanoma/metabolismo , Melanoma/secundario , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismoRESUMEN
BACKGROUND: The transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling. METHODS: We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. RESULTS: Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors. CONCLUSION: GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.
Asunto(s)
Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Factores de Transcripción de Tipo Kruppel/metabolismo , Melanoma/metabolismo , Melanoma/patología , Proteínas Nucleares/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Animales , Western Blotting , Cadherinas/metabolismo , Línea Celular Tumoral , Colágeno , Combinación de Medicamentos , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/metabolismo , Humanos , Huésped Inmunocomprometido , Inmunohistoquímica , Hibridación in Situ , Factores de Transcripción de Tipo Kruppel/genética , Laminina , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Melanoma/secundario , Ratones , Invasividad Neoplásica , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , Proteoglicanos , ARN Mensajero/metabolismo , Transducción de Señal , Regulación hacia Arriba , Proteína Gli2 con Dedos de ZincRESUMEN
Proliferative resistance to transforming growth factor beta (TGF-beta) is regarded as a critical turning point in the malignant progression of many cancer types. In melanoma this resistance is associated with more aggressive metastatic behaviour. A recent study by our group identified proliferative and invasive subtypes of melanoma cultures and found that these are, respectively, susceptible and resistant to TGF-beta suppression of proliferation. Here, using previously characterised proliferative and invasive phenotype melanoma cultures, we explored molecular responses involved in modulating susceptibility to TGF-beta-mediated inhibition of proliferation. The Id2 gene was identified as being expressed more strongly in invasive phenotype cells less susceptible to TGF-beta repression than in proliferative phenotype cells. We correlated TGF-beta repression of Id2 gene expression in proliferative phenotype cells with p15(Ink4b) induction and cell cycle arrest. Furthermore, ectopic Id2 expression in proliferative phenotype cells counteracted p15(Ink4b) induction and consequently protected them from TGF-beta-mediated inhibition of proliferation. We conclude that transition to increased aggressiveness in melanoma cells requires Id2 upregulation to suppress TGF-beta induction of p15(Ink4b) and thus help to circumvent TGF-beta-mediated inhibition of proliferation.
Asunto(s)
Proliferación Celular , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína 2 Inhibidora de la Diferenciación/fisiología , Melanoma/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Línea Celular Tumoral , Fase G1/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/patología , Invasividad NeoplásicaRESUMEN
The actin-binding protein filamin A (FLNa) affects the intracellular trafficking of various classes of receptors and has a potential role in oncogenesis. However, it is unclear whether FLNa regulates the signaling capacity and/or down-regulation of the activated epidermal growth factor receptor (EGFR). Here it is shown that partial knockdown of FLNa gene expression blocked ligand-induced EGFR responses in metastatic human melanomas. To gain greater insights into the role of FLNa in EGFR activation and intracellular sorting, we used M2 melanoma cells that lack endogenous FLNa and a subclone in which human FLNa cDNA has been stably reintroduced (M2A7 cells). Both tyrosine phosphorylation and ubiquitination of EGFR were significantly lower in epidermal growth factor (EGF)-stimulated M2 cells when compared with M2A7 cells. Moreover, the lack of FLNa interfered with EGFR interaction with the ubiquitin ligase c-Cbl. M2 cells exhibited marked resistance to EGF-induced receptor degradation, which was very active in M2A7 cells. Despite comparable rates of EGF-mediated receptor endocytosis, internalized EGFR colocalized with the lysosomal marker lysosome-associated membrane protein-1 in M2A7 cells but not M2 cells, in which EGFR was found to be sequestered in large vesicles and subsequently accumulated in punctated perinuclear structures after EGF stimulation. These results suggest the requirement of FLNa for efficient EGFR kinase activation and the sorting of endocytosed receptors into the degradation pathway.
Asunto(s)
Proteínas Contráctiles/metabolismo , Receptores ErbB/metabolismo , Melanoma/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfotransferasas/metabolismo , Transducción de Señal/fisiología , Biopsia , Línea Celular Tumoral , Proteínas Contráctiles/genética , ADN Complementario/genética , Regulación hacia Abajo/fisiología , Endocitosis/fisiología , Filaminas , Humanos , Lisosomas/metabolismo , Melanoma/patología , Proteínas de Microfilamentos/genética , Proteínas Proto-Oncogénicas c-cbl/metabolismo , TransfecciónRESUMEN
We have previously shown that Wnt5A and ROR2, an orphan tyrosine kinase receptor, interact to mediate melanoma cell motility. In other cell types, this can occur through the interaction of ROR2 with the cytoskeletal protein filamin A. Here, we found that filamin A protein levels correlated with Wnt5A levels in melanoma cells. Small interfering RNA (siRNA) knockdown of WNT5A decreased filamin A expression. Knockdown of filamin A also corresponded to a decrease in melanoma cell motility. In metastatic cells, filamin A expression was predominant in the cytoplasm, which western analysis indicated was due to the cleavage of filamin A in these cells. Treatment of nonmetastatic melanoma cells with recombinant Wnt5A increased filamin A cleavage, and this could be prevented by the knockdown of ROR2 expression. Further, BAPTA-AM chelation of intracellular calcium also inhibited filamin A cleavage, leading to the hypothesis that Wnt5A/ROR2 signaling could cleave filamin A through activation of calcium-activated proteases, such as calpains. Indeed, WNT5A knockdown decreased calpain 1 expression, and by inhibiting calpain 1 either pharmacologically or using siRNA, it decreased cell motility. Our results indicate that Wnt5A activates calpain-1, leading to the cleavage of filamin A, which results in a remodeling of the cytoskeleton and an increase in melanoma cell motility.
Asunto(s)
Calpaína/metabolismo , Proteínas Contráctiles/metabolismo , Melanoma/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas Wnt/metabolismo , Calpaína/genética , Línea Celular Tumoral , Movimiento Celular/fisiología , Quelantes/farmacología , Citoesqueleto/fisiología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Filaminas , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/patología , Melanoma/fisiopatología , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/fisiopatología , Proteínas Wnt/genética , Proteína Wnt-5aRESUMEN
Metastatic melanoma represents a complex and heterogeneous disease for which there are no therapies to improve patient survival. Recent expression profiling of melanoma cell lines identified two transcription signatures, respectively, corresponding with proliferative and invasive cellular phenotypes. A model derived from these findings predicts that in vivo melanoma cells may switch between these states. Here, DNA microarray-characterized cell lines were subjected to in vitro characterization before s.c. injection into immunocompromised mice. Tumor growth rates were measured and postexcision samples were assessed by immunohistochemistry to identify invasive and proliferative signature cells. In vitro tests showed that proliferative signature melanoma cells are faster growing but less motile than invasive signature cells. In vivo proliferative signature cells initiated tumor growth in 14 +/- 3 days postinjection. By comparison, invasive signature cells required a significantly longer (P < 0.001) period of 59 +/- 11 days. Immunohistochemistry showed that regardless of the seed cell signature, tumors showed evidence for both proliferative and invasive cell types. Furthermore, proliferative signature cell types were detected most frequently in the peripheral margin of growing tumors. These data indicate that melanoma cells undergo transcriptional signature switching in vivo likely regulated by local microenvironmental conditions. Our findings challenge previous models of melanoma progression that evoke one-way changes in gene expression. We present a new model for melanoma progression that accounts for transcription signature plasticity and provides a more rational context for explaining observed melanoma biology.