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1.
Int J Cancer ; 136(4): E161-72, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25053293

RESUMO

The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal-like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF-ß) and high expression of the stem cell marker CD44 were refractory to sorafenib-induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial-like cells expressing the stem-related proteins EpCAM or CD133 were sensitive to sorafenib-induced apoptosis both in vitro and in vivo. A cross-talk between the TGF-ß pathway and the acquisition of a mesenchymal-like phenotype with up-regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock-down in the mesenchymal-like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib-induced apoptosis. However, CD44 effect requires a TGF-ß-induced mesenchymal background, since the only overexpression of CD44 in epithelial-like HCC cells is not sufficient to impair sorafenib-induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF-ß pathway, may predict lack of response to sorafenib in HCC patients.


Assuntos
Antineoplásicos/farmacologia , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Animais , Apoptose , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/patologia , Camundongos Nus , Niacinamida/farmacologia , Fenótipo , Sorafenibe , Fator de Crescimento Transformador beta/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Cancers (Basel) ; 16(17)2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39272836

RESUMO

UM is an aggressive intraocular tumor characterized by high plasticity and a propensity to metastasize in the liver. However, the underlying mechanisms governing liver tropism remain poorly understood. Given the emerging significance of exosomes, we sought to investigate the contribution of UM-derived exosomes to specific steps of the metastatic process. Firstly, we isolated exosomes from UM cells sharing a common genetic background and different metastatic properties. A comparison of protein cargo reveals an overrepresentation of proteins related to cytoskeleton remodeling and actin filament-based movement in exosomes derived from the parental cells that may favor the detachment of cells from the primary site. Secondly, we assessed the role of macrophages in reprogramming the HHSCs by exosomes. The activation of HHSCs triggered a pro-inflammatory and pro-fibrotic environment through cytokine production, upregulation of extracellular matrix molecules, and the activation of signaling pathways. Finally, we found that activated HHSCs promote increased adhesion and migration of UM cells. Our findings shed light on the pivotal role of exosomes in pre-metastatic niche construction in the liver.

3.
Mol Oncol ; 18(10): 2471-2494, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38978350

RESUMO

Paired related homeobox 1 (PRRX1) is an inducer of epithelial-to-mesenchymal transition (EMT) in different types of cancer cells. We detected low PRRX1 expression in nevus but increased levels in primary human melanoma and cell lines carrying the BRAFV600E mutation. High expression of PRRX1 correlates with invasiveness and enrichment of genes belonging to the EMT programme. Conversely, we found that loss of PRRX1 in metastatic samples is an independent prognostic predictor of poor survival for melanoma patients. Here, we show that stable depletion of PRRX1 improves the growth of melanoma xenografts and increases the number of distant spontaneous metastases, compared to controls. We provide evidence that loss of PRRX1 counteracts the EMT phenotype, impairing the expression of other EMT-related transcription factors, causing dysregulation of the ERK and signal transducer and activator of transcription 3 (STAT3) signaling pathways, and abrogating the invasive and migratory properties of melanoma cells while triggering the up-regulation of proliferative/melanocytic genes and the expression of the neural-crest-like markers nerve growth factor receptor (NGFR; also known as neurotrophin receptor p75NTR) and neural cell adhesion molecule L1 (L1CAM). Overall, our results indicate that loss of PRRX1 triggers a switch in the invasive programme, and cells de-differentiate towards a neural crest stem cell (NCSC)-like phenotype that accounts for the metastatic aggressiveness.


Assuntos
Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio , Melanoma , Metástase Neoplásica , Humanos , Melanoma/genética , Melanoma/patologia , Melanoma/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Prognóstico , Linhagem Celular Tumoral , Animais , Transição Epitelial-Mesenquimal/genética , Inativação Gênica , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Camundongos , Movimento Celular/genética , Invasividade Neoplásica/genética , Feminino , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas do Tecido Nervoso , Receptores de Fator de Crescimento Neural
4.
Melanoma Res ; 33(1): 1-11, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36302215

RESUMO

Uveal melanoma is the most common intraocular malignancy in adults. Despite the effective primary treatment, up to 50% of patients with uveal melanoma will develop metastatic lesions mainly in the liver, which are resistant to conventional chemotherapy and lead to patient's death. To date, no orthotopic murine models of uveal melanoma which can develop spontaneous metastasis are available for preclinical studies. Here, we describe a spontaneous metastatic model of uveal melanoma based on the orthotopic injection of human uveal melanoma cells into the suprachoroidal space of immunodeficient NSG mice. All mice injected with bioluminescent OMM2.5 ( n = 23) or MP41 ( n = 19) cells developed a primary tumor. After eye enucleation, additional bioluminescence signals were detected in the lungs and in the liver. At necropsy, histopathological studies confirmed the presence of lung metastases in 100% of the mice. Liver metastases were assessed in 87 and in 100% of the mice that received OMM2.5 or MP41 cells, respectively. All tumors and metastatic lesions expressed melanoma markers and the signaling molecules insulin-like growth factor type I receptor and myristoylated alanine-rich C-kinase substrate, commonly activated in uveal melanoma. The novelty of this orthotopic mouse xenograft model is the development of spontaneous metastases in the liver from the primary site, reproducing the organoespecificity of metastasis observed in uveal melanoma patients. The faster growth and the high metastatic incidence may be attributed at least in part, to the severe immunodeficiency of NSG mice. This model may be useful for preclinical testing of targeted therapies with potential uveal melanoma antimetastatic activity and to study the mechanisms involved in liver metastasis.


Assuntos
Neoplasias Hepáticas , Melanoma , Neoplasias Cutâneas , Neoplasias Uveais , Adulto , Humanos , Animais , Camundongos , Melanoma/patologia , Xenoenxertos , Modelos Animais de Doenças , Neoplasias Uveais/patologia , Neoplasias Hepáticas/secundário
5.
J Biol Chem ; 286(2): 1475-85, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21078678

RESUMO

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.


Assuntos
Receptores ErbB/metabolismo , Receptores de Hialuronatos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Versicanas/metabolismo , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Receptores ErbB/genética , Humanos , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Isomerismo , Melanoma/patologia , Estrutura Terciária de Proteína , RNA Interferente Pequeno , Neoplasias Cutâneas/patologia , Versicanas/química , Versicanas/genética
6.
Nanomedicine ; 8(7): 1208-15, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22206945

RESUMO

Lung cancer remains a leading cause of death due to the low efficacy of chemotherapy, mainly related to the administration route used. Therefore, alternative administration routes are needed. Paclitaxel (PTX) is an insoluble anticancer drug active against solid tumors, such as those found in lung cancer, that has stimulated an intense research effort over recent years. Solid lipid nanoparticles (SLNs) are potential carriers for poorly soluble drugs, being biodegradable systems that served as alternatives to the usual colloidal carriers. That system was used to deliver PTX to the lungs and seem to fulfill the requirements for an optimum particulate carrier. Furthermore, PTX-loaded SLN pulmonary administration provided a target administration, which is expected to avoid high concentration of the drug at nontarget tissues, reducing toxicity, and increasing the drug's therapeutic index. The rationale of this study was to deliver a colloidal system to the lung lymphatics through a pulmonary route for cancer therapy. FROM THE CLINICAL EDITOR: Paclitaxel-loaded solid lipid nanoparticles were used to target tumors in a murine lung cancer model enabling high PTX concentration in the target with reduced systemic toxicity and increased therapeutic index.


Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Portadores de Fármacos/química , Lipídeos/química , Neoplasias Pulmonares/tratamento farmacológico , Pulmão/efeitos dos fármacos , Paclitaxel/administração & dosagem , Administração por Inalação , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Nebulizadores e Vaporizadores , Paclitaxel/uso terapêutico , Ratos
7.
Pigment Cell Melanoma Res ; 35(4): 450-460, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35587097

RESUMO

We recently reported an RAF rearrangement without NRAS or BRAF mutations in lesions from Giant Congenital Melanocytic Nevi (CMN). The new gene fusion involves the 5'-end of the promoter-containing N terminus of the SOX5 gene fused to exons 7-16 of the 3'-end of RAF1 gene leading to a SOX5-RAF1 fusion transcript which loses the auto-inhibitory CR1 domain but retains the complete in-frame coding sequence for the C-Terminal kinase domain of the RAF1. Stable expression of SOX5-RAF1 fusion induced growth factor-independent cell growth in murine hematopoietic Ba/F3 cells and melan-a immortalized melanocytes. Besides, it led to the transformation of both Ba/F3 and NIH 3T3 cells as revealed by colony formation assays. Furthermore, its expression results in MAPK activation assessed by increased levels of p-ERK protein in the cytosol of transduced cells. Treatment with Sorafenib and UO126 inhibited proliferation of Ba/F3-SOX5-RAF1 cells in the absence of IL3 but not the PLX 4720, a specific inhibitor of BRAF. Moreover, the tumorigenic and metastatic capacities of SOX5-RAF1 were assessed in vivo. These results indicate that SOX5-RAF1, a driver event for CMN development, has oncogenic capacity. Thus, sequencing of CMN transcriptomes may lead to the identification of this druggable fusion and interfere with the progression toward melanoma.


Assuntos
Sistema de Sinalização das MAP Quinases , Nevo Pigmentado , Proteínas Proto-Oncogênicas c-raf/genética , Fatores de Transcrição SOXD/genética , Neoplasias Cutâneas , Animais , Fusão Gênica , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Nevo Pigmentado/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/patologia
8.
Cancers (Basel) ; 14(5)2022 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-35267510

RESUMO

Malignant melanoma is a highly aggressive tumor causing most skin cancer-related deaths. Understanding the fundamental mechanisms responsible for melanoma progression and therapeutic evasion is still an unmet need for melanoma patients. Progression of skin melanoma and its dissemination to local or distant organs relies on phenotypic plasticity of melanoma cells, orchestrated by EMT-TFs and microphthalmia-associated TF (MITF). Recently, melanoma phenotypic switching has been proposed to uphold context-dependent intermediate cell states benefitting malignancy. LOXL3 (lysyl oxidase-like 3) promotes EMT and has a key role in human melanoma cell survival and maintenance of genomic integrity. To further understand the role of Loxl3 in melanoma, we generated a conditional Loxl3-knockout (KO) melanoma mouse model in the context of BrafV600E-activating mutation and Pten loss. Melanocyte-Loxl3 deletion increased melanoma latency, decreased tumor growth, and reduced lymph node metastatic dissemination. Complementary in vitro and in vivo studies in mouse melanoma cells confirmed Loxl3's contribution to melanoma progression and metastasis, in part by modulating phenotypic switching through Snail1 and Prrx1 EMT-TFs. Importantly, a novel LOXL3-SNAIL1-PRRX1 axis was identified in human melanoma, plausibly relevant to melanoma cellular plasticity. These data reinforced the value of LOXL3 as a therapeutic target in melanoma.

9.
Pigment Cell Melanoma Res ; 31(4): 484-495, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29385656

RESUMO

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).


Assuntos
Canais de Cálcio Tipo T/metabolismo , Movimento Celular , Melanoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Substituição de Aminoácidos , Canais de Cálcio Tipo T/genética , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Melanoma/patologia , Proteínas Associadas aos Microtúbulos/genética , Invasividade Neoplásica , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Transcrição da Família Snail/genética
10.
Cancer Lett ; 392: 39-50, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28161507

RESUMO

As part of its potential pro-tumorigenic actions, Transforming Growth Factor-(TGF)-ß induces epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells. Whether EMT induces changes in tumor cell plasticity has not been fully explored yet. Here, we analyze the effects of TGF-ß on the EMT and stem-related properties of HCC cells and the potential correlation among those processes. The translational aim of the study was to propose a TGF-ß/EMT/stem gene signature that would help in recognizing HCC patients as good candidates for anti-TGF-ß therapy. Results indicate that when TGF-ß induces EMT in HCC cells, a switch in the expression of stem genes is observed and their stemness potential and migratory/invasive capacity are enhanced. However, TGF-ß may induce a partial EMT in some epithelial HCC cells, increasing the expression of mesenchymal genes and CD44, but maintaining epithelial gene expression. Epithelial cells show higher stemness potential than the mesenchymal ones, but respond to TGF-ß increasing their migratory and invasive capacity. In HCC patient samples, TGFB1 expression most frequently correlates with a partial EMT, increase in mesenchymal genes and CD44 expression, as well as maintenance or over-expression of epithelial-related genes.


Assuntos
Carcinoma Hepatocelular/metabolismo , Movimento Celular , Plasticidade Celular , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Hepáticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Plasticidade Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcriptoma , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia
11.
J Invest Dermatol ; 137(6): 1297-1310, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28188776

RESUMO

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.


Assuntos
Aspartato-tRNA Ligase/genética , Aurora Quinase A/genética , Proteína Forkhead Box M1/metabolismo , Regulação Neoplásica da Expressão Gênica , Mutação , Aminoacil-RNA de Transferência/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Humanos , Melanoma/genética , Melanoma/patologia , Melanoma/terapia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Sensibilidade e Especificidade , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Int J Biochem Cell Biol ; 38(4): 544-62, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16343978

RESUMO

We analyzed the differential gene expression between variants of MDA-MB-435 human breast cancer cell line that share an identical genetic background but have different metastatic ability. The major histocompatibility complex class II was found down-regulated in highly metastatic cells and correlated with MHC transactivator (CIITA) expression. Constitutive CIITA expression observed in poorly metastatic is driven by promoters III and IV of CIITA gene. Conversely, both promoters were ineffective in highly metastatic cells. The MHC class II and CIITA expression was restored in these cells upon stimulation with IFNgamma or by the treatment with a hypomethylating agent. Both treatments induced USF-1 and IRF binding complexes to promoter IV but only IFNgamma induced the binding of 435-Lung2 nuclear proteins to an ARE-1 site at the promoter III. Neither Southern blot nor bisulfite sequencing of promoter IV demonstrated strong hypermethylation of this promoter at the IFNgamma-responsive elements such as GAS, E-box or IRF-1. We suggest that partial or hemimethylation of promoter IV is sufficient to silence the CIITA expression in highly metastatic cells and that this epigenetic mechanism is responsible for the lack of MHC-II expression. Forced CIITA expression restored the MHC-II antigen expression in 435-Lung2 cells and abrogates spontaneous lung metastasis in both SCID and nude mice but also affected the tumorigenicity in nude mice. The increase in NK cell infiltration in nude mice bearing CIITA-tumors correlated with sign of tumor cell apoptosis and the increase in the number of NK cells in the spleens, suggesting that NK cells might be responsible for the observed antitumor activity.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/biossíntese , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Apoptose/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica/genética , Metástase Neoplásica/imunologia , Metástase Neoplásica/patologia , Proteínas de Neoplasias/imunologia , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/imunologia , Elementos de Resposta/imunologia , Transativadores/imunologia
13.
Oncol Rep ; 14(5): 1337-43, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16211306

RESUMO

In the protein-targeted therapy for cancer, transferrin (Tf) is used to reach a selective and specific target in cancer cells. Tf is used conjugated to chemotherapeutic drugs, insulin, toxins, antibodies, polymers, nanoparticles, lipoplexes and liposomes. Using this latter approach, hydrophobically derivatized Tf was incorporated to liposomal bilayers. The biological activity of Tf-liposome was tested using MXT-B2 cells, a metastatic mammary carcinoma cell line. In Tf binding assays, the Scatchard analysis indicated 4.5x10(5) Tf receptors/cell. In cell growth assays, Tf-liposomes stimulated cell growth in a dose-dependent manner, up to a maximum of 32% of the total free Tf stimulation. Following this, we prepared Tf-liposomes encapsulating adriamycin (ADR) at two different ADR-to-lipid ratios. In vitro cytotoxicity assays against MXT-B2 cells gave IC(50) values 2.1-times lower for Tf-liposomal ADR in comparison to control liposomal ADR. However, similar IC(50) values were found for low ADR-to-lipid ratio Tf-liposomal ADR, as well as for control liposomal ADR. The free Tf added in excess increased the IC(50) value of Tf-liposomal ADR by 51%, while the IC(50) value of control liposomal ADR was unaffected, supporting a receptor-mediated mechanism of targeting by Tf. In addition, the lower IC(50) value is correlated with a higher total of ADR accumulation in the cells.


Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Carcinoma Papilar/patologia , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Neoplasias Mamárias Animais/patologia , Transferrina/química , Animais , Antibióticos Antineoplásicos/farmacologia , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Lipídeos , Lipossomos , Camundongos , Metástase Neoplásica , Células Tumorais Cultivadas
14.
Nat Commun ; 6: 7935, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26228240

RESUMO

Coordinated activity of VEGF and Notch signals guides the endothelial cell (EC) specification into tip and stalk cells during angiogenesis. Notch activation in stalk cells leads to proliferation arrest via an unknown mechanism. By using gain- and loss-of-function gene-targeting approaches, here we show that PTEN is crucial for blocking stalk cell proliferation downstream of Notch, and this is critical for mouse vessel development. Endothelial deletion of PTEN results in vascular hyperplasia due to a failure to mediate Notch-induced proliferation arrest. Conversely, overexpression of PTEN reduces vascular density and abrogates the increase in EC proliferation induced by Notch blockade. PTEN is a lipid/protein phosphatase that also has nuclear phosphatase-independent functions. We show that both the catalytic and non-catalytic APC/C-Fzr1/Cdh1-mediated activities of PTEN are required for stalk cells' proliferative arrest. These findings define a Notch-PTEN signalling axis as an orchestrator of vessel density and implicate the PTEN-APC/C-Fzr1/Cdh1 hub in angiogenesis.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdh1/metabolismo , Proliferação de Células/genética , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/metabolismo , Receptores Notch/metabolismo , Animais , Imunofluorescência , Immunoblotting , Camundongos , PTEN Fosfo-Hidrolase/metabolismo , Reação em Cadeia da Polimerase
15.
J Invest Dermatol ; 123(6): 1151-61, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15610528

RESUMO

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.


Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Melanoma , Neoplasias Cutâneas , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Cinética , Neovascularização Patológica/fisiopatologia , Fosfatidilinositol 3-Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
16.
Eur J Pharm Sci ; 18(3-4): 221-9, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12659933

RESUMO

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 microm-microspheres. These systems exhibit a similar pattern of release that is characterized by an initial burst (2-24% IFN-alpha 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.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Interferon-alfa/administração & dosagem , Interferon-alfa/farmacocinética , Microesferas , Nanotubos , Biodegradação Ambiental , Linhagem Celular Tumoral , Humanos , Ácido Láctico/administração & dosagem , Ácido Láctico/farmacocinética , Ácido Poliglicólico/administração & dosagem , Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/administração & dosagem , Polímeros/farmacocinética
17.
Oncotarget ; 5(6): 1439-51, 2014 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-24742402

RESUMO

Germline mutations in CDKN2A and/or red hair color variants in MC1R genes are associated with an increased susceptibility to develop cutaneous melanoma or non melanoma skin cancer. We studied the impact of the CDKN2A germinal mutation p.G101W and MC1R variants on gene expression and transcription profiles associated with skin cancer. To this end we set-up primary skin cell co-cultures from siblings of melanoma prone-families that were later analyzed using the expression array approach. As a result, we found that 1535 transcripts were deregulated in CDKN2A mutated cells, with over-expression of immunity-related genes (HLA-DPB1, CLEC2B, IFI44, IFI44L, IFI27, IFIT1, IFIT2, SP110 and IFNK) and down-regulation of genes playing a role in the Notch signaling pathway. 3570 transcripts were deregulated in MC1R variant carriers. In particular, genes related to oxidative stress and DNA damage pathways were up-regulated as well as genes associated with neurodegenerative diseases such as Parkinson's, Alzheimer and Huntington. Finally, we observed that the expression signatures indentified in phenotypically normal cells carrying CDKN2A mutations or MC1R variants are maintained in skin cancer tumors (melanoma and squamous cell carcinoma). These results indicate that transcriptome deregulation represents an early event critical for skin cancer development.


Assuntos
Biomarcadores Tumorais/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Predisposição Genética para Doença , Melanoma/genética , Mutação/genética , Receptor Tipo 1 de Melanocortina/genética , Neoplasias Cutâneas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Células Cultivadas , Técnicas de Cocultura , Perfilação da Expressão Gênica , Genótipo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/patologia , Melanoma Maligno Cutâneo
18.
PLoS One ; 8(10): e76247, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098452

RESUMO

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3'-UTRs of ENPEP, CK2-α, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-α, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40-56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-α was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-α, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-α, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.


Assuntos
Neoplasias da Mama/genética , Caseína Quinase II/genética , Transformação Celular Neoplásica/genética , Ciclinas/genética , Genes Supressores de Tumor , Glutamil Aminopeptidase/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Regiões 3' não Traduzidas , Neoplasias da Mama/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular , Proliferação de Células , Análise por Conglomerados , Ciclinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glutamil Aminopeptidase/metabolismo , Humanos , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA
19.
Cancer Cell ; 22(6): 709-24, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23201163

RESUMO

The epithelial-mesenchymal transition (EMT) is required in the embryo for the formation of tissues for which cells originate far from their final destination. Carcinoma cells hijack this program for tumor dissemination. The relevance of the EMT in cancer is still debated because it is unclear how these migratory cells colonize distant tissues to form macrometastases. We show that the homeobox factor Prrx1 is an EMT inducer conferring migratory and invasive properties. The loss of Prrx1 is required for cancer cells to metastasize in vivo, which revert to the epithelial phenotype concomitant with the acquisition of stem cell properties. Thus, unlike the classical EMT transcription factors, Prrx1 uncouples EMT and stemness, and is a biomarker associated with patient survival and lack of metastasis.


Assuntos
Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , Proteínas de Homeodomínio/fisiologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Estudos Retrospectivos , Células-Tronco/metabolismo , Células-Tronco/patologia
20.
Melanoma Res ; 21(3): 217-22, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21467954

RESUMO

Uveal melanoma metastases develop in 6.5-35% of patients, most commonly to the liver. Metastatic uveal melanoma (MUM) survival is poor, with 5-7 months of median survival. We reviewed retrospectively all patients with MUM diagnosed between January 1990 and December 2008 at our institution. We analyzed a total of 58 patients with a median age of 61 years (31-84 years). Median time for metastases development was 25.63 months (0.17-102.43 months). Fifty-six patients had hepatic involvement, 63.8% bilobar and 51.7% had more than or equal to five hepatic metastatic lesions. Sixteen patients (27.6%) had two or more organs involved. Six patients (10.71%) were treated with surgery, 25 patients (44.67%) received systemic chemotherapy, and 23 (41.07%) had best supportive care (BSC). The median overall survival (OS) for all the patients was 10.83 months [95% confidence interval (CI): 6.92-14.74]. Patients who had undergone chemotherapy presented 10.83 months (95% CI: 5.35-16.308) of median OS whereas the patients who did not undergo this treatment had an OS of 8.033 months (95% CI: 2.46-13.61). There were more patients with poor survival characteristics such as worse Eastern Cooperative Oncology Group performance status in the BSC group. OS was poor in treated and BSC patients. Differences in survival are more likely to be related to patient characteristics rather than to a chemotherapy effect. Patients with MUM should be included in clinical trials evaluating other options with newer agents.


Assuntos
Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Melanoma/tratamento farmacológico , Melanoma/patologia , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Resultado do Tratamento
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