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The cell cycle-related genes AURKA and FOXM1 are overexpressed in melanoma. We show here that AURKA overexpression is associated with poor prognosis in three independent cohorts of melanoma patients and correlates with the presence of genomic amplification of AURKA locus and BRAFV600E mutation. AURKA overexpression may also be driven by increased promoter activation through elements such as ETS and FOXM1 found within the 5′ proximal promoter region. Activated MAPK/ERK signaling pathway mediates robust AURKA promoter activation, thereby knockdown of BRAFV600E and ERK inhibition results in reduced AURKA transcription and expression. We show a positive correlation between FOXM1 and AURKA expression in three independent cohorts of melanoma patients. FOXM1 silencing decreases expression of AURKA and late cell cycle genes in melanoma cells. We further found that FOXM1 expression levels are significantly higher in tumors carrying the BRAFV600E mutation compared with the wild-type BRAF (BRAFwt). Accordingly, the knockdown of BRAFV600E also reduces the expression of FOXM1 in BRAFV600E cells. Moreover, Aurora kinase A and FOXM1 inhibition by either genetic knockdown or pharmacologic inhibitors impair melanoma growth and survival both in culture and in vivo, underscoring their therapeutic value for melanoma patients who fail to benefit from BRAF/MEK signaling inhibition.

Resumen Introduccion y objetivos Algunas formas de cancer tienden a producir metastasis en determinados organos. En cuanto al melanoma, el melanoma uveal produce metastasis casi exclusivamente en el higado, mientras que el melanoma cutaneo se disemina tambien a otros organos. A pesar de importantes avances en el conocimiento de las bases moleculares del melanoma, hay pocos estudios recientes sobre el patron de diseminacion visceral del melanoma cutaneo. Nuestro objetivo fue analizar retrospectivamente una posible asociacion entre tipo clinicopatologico y localizacion del melanoma cutaneo con el patron de diseminacion visceral y su cronologia. Material y metodos Se incluyeron en el estudio los pacientes diagnosticados de melanoma cutaneo entre 1988-2009 con mas de 2 anos de seguimiento. Resultados De un total de 1.083 pacientes 92 desarrollaron metastasis viscerales: 21 en el sistema nervioso central (SNC), 24 en los pulmones, 17 en el higado, 7 en el tubo digestivo y 23 en multiples organos simultaneamente. Las recidivas en el pulmon, el higado y el tubo digestivo se produjeron mayoritariamente antes de los 5 anos, mientras que las metastasis al SNC y a multiples organos simultaneamente fueron mas tardias (38 y 43% despues de los 5 anos respectivamente). Conclusiones A diferencia del melanoma ocular, el melanoma cutaneo se disemina por igual a multiples organos. No hemos detectado asociacion significativa respecto al organo diana de las metastasis segun el tipo histologico y tampoco segun la localizacion del tumor primario. A pesar de que la mayoria de metastasis viscerales se producen antes de los 5 anos, tambien pueden producirse metastasis viscerales mas alla de los 10 anos de seguimiento.

Background and objectives: Few studies have addressed cutaneous recurrence of melanoma. The aim of this retrospective study was to analyze the characteristics and prognostic significance of the different patterns of cutaneous recurrence. Material and methods: Patients diagnosed with melanoma between 1988 and 2008 at Hospital de Bellvitge, Barcelona, Spain and for whom data were available for at least 2 years of follow-up were included in the study. Local recurrence was defined as melanoma invasion of the skin adjacent to the scar left by excision of the primary tumor, regional metastasis or recurrence as metastasis restricted to the area drained by a regional lymph node station, and distant cutaneous metastasis as metastasis occurring outside this area. The relationship between cutaneous recurrence pattern and age, sex, primary tumor site, tumor subtype, Breslow depth, and ulceration was assessed. Results: Eighty-five out of 1,080 patients (7.87%) had cutaneous recurrence. In 71 of those patients (83.53%; 27 men and 44 women; mean age, 60.68 years), this was the first indication of melanoma recurrence. Thirty-two patients had local recurrence, 32 regional metastasis, and 7 distant metastasis. Significant differences were observed in survival time from diagnosis of the primary tumor (P = .044) and from diagnosis of cutaneous recurrence (P < .001) according to the type of recurrence. Conclusions: Our results suggest that the pattern of cutaneous recurrence is prognostically significant and related to the site of the primary tumor given that the majority of local and regional recurrences occurred in primary tumors located on the lower limbs and head.

Versican is a hyaluronan-binding, extracellular chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The short V3 isoform contains the G1 and G3 terminal domains of versican that may potentially interact directly or indirectly with the hyaluronan receptor CD44 and the EGFR, respectively. We have previously described that overexpression of V3 in MeWo human melanoma cells markedly reduces tumor cell growth in vitro and in vivo. In this study we have investigated the signaling mechanism of V3 by silencing the expression of CD44 in control and V3-expressing melanoma cells. Suppression of CD44 had the same effects on cell proliferation and cell migration than those provoked by V3 expression, suggesting that V3 acts through a CD44-mediated mechanism. Furthermore, CD44-dependent hyaluronan internalization was blocked by V3 expression and CD44 silencing, leading to an accumulation of this glycosaminoglycan in the pericellular matrix and to changes in cell migration on hyaluronan. Furthermore, ERK1/2 and p38 activation after EGF treatment were decreased in V3-expressing cells suggesting that V3 may also interact with the EGFR through its G3 domain. The existence of a EGFR/ErbB2 receptor complex able to interact with CD44 was identified in MeWo melanoma cells. V3 overexpression resulted in a reduced interaction between EGFR/ErbB2 and CD44 in response to EGF treatment. Our results indicate that the V3 isoform of versican interferes with CD44 and the CD44-EGFR/ErbB2 interaction, altering the signaling pathways, such as ERK1/2 and p38 MAPK, that regulate cell proliferation and migration.

Versican is a large chondroitin sulfate proteoglycan of the extracellular matrix that is involved in a variety of cellular processes. We showed previously that versican, which is overexpressed in cutaneous melanomas as well as in premalignant lesions, contributes to melanoma progression, favoring the detachment of cells and the metastatic dissemination. Here, we investigated the transcriptional regulation of the versican promoter in melanoma cell lines with different levels of biological aggressiveness and stages of differentiation. We show that versican promoter up-regulation accounts for the differential expression levels of mRNA and protein detected in the invasive SK-mel-131 human melanoma cells. The activity of the versican promoter increased 5-fold in these cells in comparison with that measured in non-invasive MeWo melanoma cells. Several transcriptional regulatory elements were identified in the proximal promoter, including AP-1, Sp1, AP-2, and two TCF-4 sites. We show that promoter activation is mediated by the ERK/MAPK and JNK signaling pathways acting on the AP-1 site, suggesting that BRAF mutation present in SK-mel-131 cells impinge upon the up-regulation of the versican gene through signaling elicited by the ERK/MAPK pathway. This is the first time the AP-1 transcription factor family has been shown to be related to the regulation of versican expression. Furthermore, deletion of the TCF-4 binding sites caused a 60% decrease in the promoter activity in SK-mel-131 cells. These results showing that AP-1 and TCF-4 binding sites are the main regulatory regions directing versican production provide new insights into versican promoter regulation during melanoma progression.

Id-1, a member of the helix-loop-helix transcription factor family has been shown to be involved in cell proliferation, angiogenesis and invasion of many types of human cancers. We have previously shown that stable expression of E47 and Snail repressors of the E-cadherin promoter in MDCK epithelial cell line triggers epithelial mesenchymal transition (EMT) concomitantly with changes in gene expression. We show here that both factors activate the Id-1 gene promoter and induce Id-1 mRNA and protein. The upregulation of the Id-1 gene occurs through the transactivation of the promoter by the Erk/MAPK signaling pathway. Moreover, oncogenic Ras is also able to activate Id-1 promoter in MDCK cells in the absence of both E47 and Snail transcription factors. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including AP-1, Sp1 and four putative E-boxes. By EMSA, we only detected an increased binding to Sp1 and AP-1 elements in E47- and Snail-expressing cells. Binding is affected by the treatment of cells with PD 98059 MEK inhibitor, suggesting that MAPK/Erk contributes to the recruitment or assembly of proteins to Id-1 promoter. Small interfering RNA directed against Sp1 reduced Id-1 expression and the upregulation of the promoter, indicating that Sp1 is required for Id-1 induction in E47- and Snail-expressing cells. Our results provide new insights into how some target genes are activated during and/or as a consequence of the EMT triggered by both E47 and Snail transcription factors. © 2007 Elsevier Inc. All rights reserved., This work was supported by the Spanish Ministry of Science and Technology (SAF 2002-00817 and SAF 2005-03259 to AF; SAF 2004-00361 and NAN 2004-09230 C04-02 to AC); the Instituto de Salud Carlos III, RTICCC, FIS 03-C03/10 and EU (MRTN-CT-2004-005428).

Vascular endothelial growth factor (VEGF) is an important mediator of tumor-associated angiogenesis, and consequently it has been associated with metastasis. We report here that the overexpression of VEGF 165 in melanoma xenografts promotes an acceleration of tumor growth and an increase in angiogenesis as well as the spontaneous metastasis formation. In addition, VEGF receptors (VEGFR)1, VEGFR2 and neurophilin-1 are expressed in A375 melanoma cells. Forced overexpression of VEGF in these cells induces cell growth and triggers survival activity in serum-starved cultures, by a mechanism dependent on the mitogen-activating protein kinase signaling pathway. Furthermore, these effects are dependent MEK 1/2 activity. Kinase domain region-specific tyrosine kinase inhibitors dramatically reduced DNA synthesis to 20% with respect to the controls, although they did not completely suppress either the p44 or p42-phosphorylated forms of extracellular signal-regulated protein kinase. These inhibitors also provoked a decrease in Akt phosphorylation. We observed a dramatic reduction in survival after treatment with phosphatidylinositol 3′-kinase (PI3K)-specific inhibitor in the presence of specific tyrosinase inhibitors. We suggest that the overproduction of VEGF 165 concomitantly expressed with its receptors favors cell growth and survival of melanoma cells through MAPK and PI3K signaling pathways. These data support the involvement in melanoma growth and survival of a VEGF-dependent internal autocrine loop mechanism, at least in vitro .

The development of new interferon-alpha (IFN-alpha) delivery strategies is a key issue in order to simplify its administration and improve its therapeutic effects, while reducing its dose-related side effects. One of the most attractive approaches towards this aim is the encapsulation of IFN-alpha into poly(lactic-glycolic acid) (PLGA) microspheres. Nevertheless, the stability of IFN-alpha released from these microspheres has been identified as one of the most important concerns in relation to the potential of this approach. Being conscious of this problem, we have used new strategies for the encapsulation of IFN-alpha into biodegradable micro- and nanoparticles. We chose poloxamer 188 as a stabilizing agent and encapsulated IFN-alpha within PLGA/poloxamer blend microspheres prepared by an oil-in-oil solvent extraction technique and also within PLGA micro- and nanospheres containing poloxamer, prepared by the water-in-oil-in-water solvent evaporation technique. The results showed that these techniques led to the efficient encapsulation of IFN-alpha and the modulation of their particle size, ranging from nanospheres (280 nm) to 40 μm-microspheres. These systems exhibit a similar pattern of release that is characterized by an initial burst (2–24% IFN-α released, as determined by ELISA) followed by small pulses of immunoenzymatically detected IFN-alpha for up to 1 month. The maintenance of the structural integrity and bioactivity of the protein was confirmed using a cytostasis bioassay. The results showed that the antiproliferative activity of the IFN-alpha varied depending on the formulation. More specifically, PLGA/poloxamer blend microspheres were able to provide significant amounts of active IFN-alpha for up to 96 days. This new IFN-alpha delivery system opens up possibilities to improve present IFN-alpha-based therapies.

Mouse transformed keratinocytes cultured in the presence of transforming growth factor-beta1 (TGF-beta1) acquire a set of morphological and functional properties giving rise to a more motile phenotype that expresses mesenchymal markers. In this work, we present evidence showing that TGF-beta1 stimulates cellular production of MMP-9 (Gelatinase B), a metalloproteinase that plays an important role in tumoral invasion. Our results demonstrate that TGF-beta1stimulates MMP-9 production and MMP-9 promoter activity in a process that depends of the activation of the Ras-ERK1,2 MAP kinase pathway. The latter was demonstrated by cellular transfection of TGF-beta1-sensitive cells with a RasN17 mutant gene, using PD 098059, a MEK 1,2 inhibitor, and treating cells with anti-sense oligodeoxinucleotides. The enhanced MMP-9 production proved to be an important factor in the acquisition of migratory and invasive properties as shown by the use of a specific inhibitor of MMP-9 (GM6001) that inhibits the TGF-beta1-stimulated invasive and migratory properties of these transformed keratinocytes.

The aim of this paper was to evaluate the potential of chitosan nanoparticles as carriers for the anthracycline drug, doxorubicin (DOX). The challenge was to entrap a cationic, hydrophilic molecule into nanoparticles formed by ionic gelation of the positively charged polysaccharide chitosan. To achieve this objective, we attempted to mask the positive charge of DOX by complexing it with the polyanion, dextran sulfate. This modification doubled DOX encapsulation efficiency relative to controls and enabled real loadings up to 4.0 wt.% DOX. Separately, we investigated the possibility of forming a complex between chitosan and DOX prior to the formation of the particles. Despite the low complexation efficiency, no dissociation of the complex was observed upon formation of the nanoparticles. Fluorimetric analysis of the drug released in vitro showed an initial release phase, the intensity of which was dependent on the association mode, followed by a very slow release. The evaluation of the activity of DOX-loaded nanoparticles in cell cultures indicated that those containing dextran sulfate were able to maintain cytostatic activity relative to free DOX, while DOX complexed to chitosan before nanoparticle formation showed slightly decreased activity. Additionally, confocal studies showed that DOX was not released in the cell culture medium but entered the cells while remaining associated to the nanoparticles. In conclusion, these preliminary studies showed the feasibility of chitosan nanoparticles to entrap the basic drug DOX and to deliver it into the cells in its active form.

The purpose of this study was to assess the prognostic effect of the expression of E-cadherin, beta-catenin and CD44 adhesion molecules in bladder carcinoma. 22 superficial and 18 invasive bladder tumour samples were studied by immunohistochemistry. The median follow-up was 24 months (range: 1-50 months). Loss of E-cadherin and beta-catenin immunoreactivity was found in 14 (35%) and 17 (43%) tumours, respectively, and was significantly associated with invasiveness, high grade and p53 overexpression. There was no correlation between CD44 variant expression and clinicopathological findings. Loss of E-cadherin expression was an independent predictor of poor survival in a multivariate analysis, when assessed with age, grade, stage and p53 status (hazards ratio adjusted (HRa)=4.45 [95% confidence interval (CI), 1.06-18.63]). This effect was particularly augmented in patients with invasive bladder cancer. When expression of E-cadherin and beta-catenin were evaluated simultaneously, loss of immunoreactivity of both proteins was a strong predictor of poor survival (HRa=13.06 [95% CI, 0.95-178.55]). The same pattern was found when progression-free survival in relation to these variables was assessed. In conclusion, assessment of E-cadherin and beta-catenin immunoreactivity may be a useful prognostic marker in bladder cancer complementary to established prognostic factors.

10 p.-9 fig.-1 tab., Endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Endoglin glycoprotein is a component of the transforming growth factor type ß (TGF-ß) receptor system which is highly expressed by endothelial cells, and at lower levels on fibroblasts and smooth muscle cells, suggesting the involvement of these lineages in the HHT1 vascular dysplasia. Overexpression of endoglin in mouse NCTC929 fibroblasts led to decreased migration in chemotactic and wound healing assays, as well as changes in the cellular morphology. When plated on uncoated surfaces, endoglin transfectants formed intercellular clusters, endoglin being not specifically localized to the cell-cell junctions, but homogenously distributed on the cellular surface. Although the expression of α5ß1 integrin and of an activation epitope of ß1 integrin were unchanged, a polyclonal antibody to α5ß1 integrin was able to inhibit cluster formation, suggesting the involvement of integrin ligand/s. In fact, coating with fibronectin, laminin, or an RGD-containing 80 kDa fragment of fibronectin were able to prevent the cellular clustering. Furthermore, synthesis of plasminogen activator inhibitor 1 (PAI-1), and to a weak extent that of fibronectin, were inhibited in endoglin transfectants. Thus, the presence of endoglin in mouse NCTC929 fibroblasts is associated with reduced production of certain extracellular matrix (ECM) components, which might explain their altered morphology, migration and intercellular cluster formation., This work has been supported by grants from Camisión Interministerial de Ciencia yTecnología (CICYT-SAF97-0034 to C. Bernabéu, and CICYT-SAF97-0064-C03-02 to A. García-Pardo), Comunidad Autónoma de Madrid (CAM) and Biomed Program of the European Community (BMH4-CT95-0995 to C. Bernabéu).

Antecedentes e hipotesis La senalizacion del factor de crecimiento transformante-beta (TGF-β) en varios canceres humanos es un importante regulador de la transicion epitelio-mesenquimal (EMT) y puede guiar a las celulas a una de-diferenciacion de forma autocrina. Sin embargo, los mecanismos moleculares que integran el fenotipo mesenquimal con la adquisicion de propiedades de celula troncal tumoral (CSC) (tambien definida como celula iniciadora de tumores, TIC) todavia requieren una mayor investigacion. En el carcinoma hepatocelular (HCC), aun se sabe muy poco acerca de si el TGF-β regula la plasticidad celular y si eso podria estar relacionado con el proceso de EMT inducido por este factor. La hipotesis de este trabajo de tesis doctoral es que el TGF-β, que esta sobre-expresado en muchos tumores humanos de HCC, podria jugar un papel en la regulacion de EMT completa o parcial, lo que tambien podria modular la capacidad de stemness de las celulas. Objetivos El objetivo principal de este proyecto es analizar los efectos inducidos por el TGF-β sobre el fenotipo CSC y la interaccion con el programa de EMT en el carcinoma hepatocelular humano. Este objetivo se divide en cuatro objetivos mas especificos: 1. Analizar la relevancia de la via del TGF-β autocrino en la expresion de los genes de la EMT y CSC en diferentes celulas de HCC. 2. Investigar el efecto del silenciamiento de la expresion del Receptor I del TGF-β sobre el fenotipo EMT y las propiedades CSC. 3. Estudiar el efecto del tratamiento a largo plazo con el TGF-β en el fenotipo EMT y las propiedades stemness de celulas epiteliales de HCC. 4. Explorar las correlaciones in vivo entre los genes de TGF-β, stemness y EMT. Analisis de: I) los tumores de ratones con carcinogenesis hepatica inducida por la dietil-nitrosamina (DEN); II) tejidos humanos de HCC. Resultados/Conclusiones • La sobre-activacion de la via de TGF-β se correlaciona con un fenotipo mesenquimal, con mayores capacidades migratorias e invasivas y con la expresion de los marcadores CD90 y CD44. • En celulas con fenotipo plenamente mesenquimal, el TGF-β es critico no solo para la expresion de CD44 y las capacidades migratorias e invasivas, sino tambien para el control de las funciones relacionadas con stemness. • CD44 juega un papel crucial en la determinacion no solo del fenotipo mesenquimal y las habilidades tumorigenicas relacionadas con stemness, sino tambien en el potencial migratorio e invasivo de las celulas mesenquimales. • La estimulacion cronica de la via de TGF-β en las celulas Hep3B se correlaciona con un cambio en la expresion de genes epiteliales (EPCAM, CD133) por marcadores mesenquimales (CD44) relacionados con stemness, que confieren una ventaja tanto en el mantenimiento de las propiedades stemness como en una mejor capacidad para migrar e invadir. • En contraste con lo observado en las celulas Hep3B, la sobre-activacion de la via del TGF-β en celulas PLC/PRF/5 induce la expresion simultanea de EPCAM, CD133 y CD44, asi como un aumento en migracion e invasion. • Ademas, las celulas TβT-PLCCD44+ expresan genes epiteliales y mesenquimales, que muestran propiedades stemness similares a la mas epiteliales TβT-PLCCD44- pero una mucho mas alta capacidad migratoria e invasiva. • La hepatocarcinogenesis inducida por DEN en ratones coincide con aumento de TGF-β, una mezcla de genotipo epitelial/mesenquimal y la expresion simultanea de genes epiteliales y mesenquimales relacionados con stemness. • En tejidos tumorales de pacientes de HCC, la expresion de TGF-β se relaciona tanto con el fenotipo EMT (con la expresion predominante de genes mesenquimales), como con EMT parcial (con presencia simultanea de genes epiteliales y mesenquimales). Sin embargo, la firma genetica EMT/stemness mas frecuente coincide con la sobre regulacion de la expresion de TGF-β y con EMT parcial, lo que lleva a una expresion concomitante de genes epiteliales y mesenquimales relacionados con stemness.

We examined whether fibroblasts from subcutaneous, colon or lung tissues of nude mice influence the invasive potential of highly metastatic human colon carcinoma KM12SM cells. Primary cultures of nude mouse fibroblasts from skin, lung and colon were established. Invasive and metastatic KM12SM cells were cultured alone or with fibroblasts. Growth and invasive properties of the KM12SM cells were evaluated as well as their production of gelatinase activity. KM12SM cells were able to grow on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts. The conditioned media of KM12SM cells cocultured with skin, colon or lung fibroblasts were examined for the presence of type IV collagenase (gelatinase). KM12SM growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of 64 kDa type IV collagenase, whereas KM12SM cells cocultivated with nude mouse skin fibroblasts did not. In contrast, human squamous cell carcinoma A431 cells produced significant levels of collagenase type IV when cocultured with nude mouse skin fibroblasts, a tissue they invaded and completely penetrated. Incubation of KM12SM cells in serum-free medium containing recombinant human interferon-beta (fibroblast interferon) was associated with significant reduction in gelatinase activity. Since the production of type IV collagenase by human colon cancer cells is specifically inhibited by mouse skin fibroblasts but not by colon or lung fibroblasts the data suggest that organ-specific fibroblasts can influence the invasive and metastatic properties of KM12SM cells.