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1.
Biology (Basel) ; 10(9)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34571806

RESUMO

Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.

2.
J Exp Clin Cancer Res ; 40(1): 228, 2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253243

RESUMO

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, characterized by a poor prognosis mainly due to recurrence and therapeutic resistance. It has been widely demonstrated that glioblastoma stem-like cells (GSCs), a subpopulation of tumor cells endowed with stem-like properties is responsible for tumor maintenance and progression. Moreover, it has been demonstrated that GSCs contribute to GBM-associated neovascularization processes, through different mechanisms including the transdifferentiation into GSC-derived endothelial cells (GdECs). METHODS: In order to identify druggable cancer-related pathways in GBM, we assessed the effect of a selection of 349 compounds on both GSCs and GdECs and we selected elesclomol (STA-4783) as the most effective agent in inducing cell death on both GSC and GdEC lines tested. RESULTS: Elesclomol has been already described to be a potent oxidative stress inducer. In depth investigation of the molecular mechanisms underlying GSC and GdEC response to elesclomol, confirmed that this compound induces a strong increase in mitochondrial reactive oxygen species (ROS) in both GSCs and GdECs ultimately leading to a non-apoptotic copper-dependent cell death. Moreover, combined in vitro treatment with elesclomol and the alkylating agent temozolomide (TMZ) enhanced the cytotoxicity compared to TMZ alone. Finally, we used our experimental model of mouse brain xenografts to test the combination of elesclomol and TMZ and confirmed their efficacy in vivo. CONCLUSIONS: Our results support further evaluation of therapeutics targeting oxidative stress such as elesclomol with the aim of satisfying the high unmet medical need in the management of GBM.

3.
Hum Antibodies ; 29(1): 63-84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33164927

RESUMO

BACKGROUND: The NCAM or CD56 antigen is a cell surface glycoprotein belonging to the immunoglobulin super-family involved in cell-cell and cell-matrix adhesion. NCAM is also over-expressed in many tumour types and is considered a tumour associated antigen, even if its role and biological mechanisms implicated in tumour progression and metastasis have not yet to be elucidated. In particular, it is quite well documented the role of the interaction between the NCAM protein and the fibroblast growth factor receptor-1 in metastasis and invasion, especially in the ovarian cancer progression. OBJECTIVE: Here we describe the isolation and preliminary characterization of a novel human anti-NCAM single chain Fragment variable antibody able to specifically bind NCAM-expressing cells, including epithelial ovarian cancer cells. METHODS: The antibody was isolate by phage display selection and was characterized by ELISA, FACS analysis and SPR experiments. Interference in EOC migration was analyzed by scratch test. RESULTS: It binds a partially linear epitope lying in the membrane proximal region of two fibronectin-like domains with a dissociation constant of 3.43 × 10-8 M. Interestingly, it was shown to interfere with the NCAM-FGFR1 binding and to partially decrease migration of EOC cells. CONCLUSIONS: According to our knowledge, this is the first completely human antibody able to interfere with this newly individuated cancer mechanism.


Assuntos
Bacteriófagos , Transdução de Sinais , Bacteriófagos/metabolismo , Humanos , Imunoglobulinas , Moléculas de Adesão de Célula Nervosa/metabolismo , Ligação Proteica
4.
Cancer Res ; 80(19): 4087-4102, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32718996

RESUMO

Cancer stem-like cells (CSC) induce aggressive tumor phenotypes such as metastasis formation, which is associated with poor prognosis in triple-negative breast cancer (TNBC). Repurposing of FDA-approved drugs that can eradicate the CSC subcompartment in primary tumors may prevent metastatic disease, thus representing an effective strategy to improve the prognosis of TNBC. Here, we investigated spheroid-forming cells in a metastatic TNBC model. This strategy enabled us to specifically study a population of long-lived tumor cells enriched in CSCs, which show stem-like characteristics and induce metastases. To repurpose FDA-approved drugs potentially toxic for CSCs, we focused on pyrvinium pamoate (PP), an anthelmintic drug with documented anticancer activity in preclinical models. PP induced cytotoxic effects in CSCs and prevented metastasis formation. Mechanistically, the cell killing effects of PP were a result of inhibition of lipid anabolism and, more specifically, the impairment of anabolic flux from glucose to cholesterol and fatty acids. CSCs were strongly dependent upon activation of lipid biosynthetic pathways; activation of these pathways exhibited an unfavorable prognostic value in a cohort of breast cancer patients, where it predicted high probability of metastatic dissemination and tumor relapse. Overall, this work describes a new approach to target aggressive CSCs that may substantially improve clinical outcomes for patients with TNBC, who currently lack effective targeted therapeutic options. SIGNIFICANCE: These findings provide preclinical evidence that a drug repurposing approach to prevent metastatic disease in TNBC exploits lipid anabolism as a metabolic vulnerability against CSCs in primary tumors.


Assuntos
Antineoplásicos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Compostos de Pirvínio/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colesterol/metabolismo , Reposicionamento de Medicamentos , Feminino , Glucose/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos Endogâmicos NOD , Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Mol Sci ; 21(10)2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32443824

RESUMO

Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neurais/metabolismo , Adulto , Animais , Neoplasias Encefálicas/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Células HEK293 , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células Tumorais Cultivadas
6.
Neuro Oncol ; 22(12): 1771-1784, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-32459347

RESUMO

BACKGROUND: Glioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene‒type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis. METHODS: Real-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3). RESULTS: Loss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan-Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT). CONCLUSION: In GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.


Assuntos
Glioblastoma , RNA Longo não Codificante , Adulto , Proteínas de Ligação ao Cálcio , Proliferação de Células , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Impressão Genômica , Glioblastoma/genética , Humanos , Proteínas de Membrana/genética , RNA Longo não Codificante/genética
7.
Int J Mol Sci ; 21(7)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244500

RESUMO

MicroRNAs are tiny but powerful regulators of gene expression at the post-transcriptional level. Aberrant expression of oncogenic and tumor-suppressor microRNAs has been recognized as a common feature of human cancers. Colorectal cancer represents a major clinical challenge in the developed world and the design of innovative therapeutic approaches relies on the identification of novel biological targets. Here, we perform a functional screening in colorectal cancer cells using a library of locked nucleic acid (LNA)-modified anti-miRs in order to unveil putative oncogenic microRNAs whose inhibition yields a cytotoxic effect. We identify miR-1285-3p and further explore the effect of its targeting in both commercial cell lines and primary colorectal cancer stem cells, finding induction of cell cycle arrest and apoptosis. We show that DAPK2, a known tumor-suppressor, is a novel miR-1285 target and mediates both the anti-proliferative and the pro-apoptotic effects of miR-1285 depletion. Altogether, our findings uncover a novel oncogenic microRNA in colorectal cancer and lay the foundation for further studies aiming at the development of possible therapeutic strategies based on miR-1285 targeting.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Proteínas Quinases Associadas com Morte Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas , Oligonucleotídeos
8.
J Exp Clin Cancer Res ; 39(1): 2, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910865

RESUMO

BACKGROUND: Quiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown. METHODS: A population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets. RESULTS: Untreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes. CONCLUSIONS: These results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.


Assuntos
Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Neoplásicas/metabolismo , Regulação para Cima , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Oxaliplatina/farmacologia , Prognóstico
9.
Cancers (Basel) ; 12(1)2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861603

RESUMO

The question whether perivascular glioma cells invading the brain far from the tumor bulk may disrupt the blood-brain barrier (BBB) represents a crucial issue because under this condition tumor cells would be no more protected from the reach of chemotherapeutic drugs. A recent in vivo study that used human xenolines, demonstrated that single glioma cells migrating away from the tumor bulk are sufficient to breach the BBB. Here, we used brain xenografts of patient-derived glioma stem-like cells (GSCs) to show by immunostaining that in spite of massive perivascular invasion, BBB integrity was preserved in the majority of vessels located outside the tumor bulk. Interestingly, the tumor cells that invaded the brain for the longest distances traveled along vessels with retained BBB integrity. In surgical specimens of malignant glioma, the area of brain invasion showed several vessels with preserved BBB that were surrounded by tumor cells. On transmission electron microscopy, the cell inter-junctions and basal lamina of the brain endothelium were preserved even in conditions in which the tumor cells lay adjacently to blood vessels. In conclusion, BBB integrity associates with extensive perivascular invasion of glioma cells.

10.
Cell Death Dis ; 10(7): 529, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31332161

RESUMO

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.


Assuntos
Antineoplásicos/uso terapêutico , Fenretinida/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Mol Oncol ; 13(9): 1836-1854, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31115969

RESUMO

Emerging data support the rationale of combined therapies in advanced melanoma. Specifically, the combined use of drugs with different mechanisms of action can reduce the probability of selecting resistant clones. To identify agents active against melanoma cells, we screened a library of 349 anti-cancer compounds, currently in clinical use or trials, and selected PIK-75, an inhibitor of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, as the 'top active' drug. PIK-75 was then used alone or in combination with vemurafenib, the first BRAF inhibitor approved for patients with melanoma harboring BRAF mutations. We identified a combined dose of PIK-75 and vemurafenib that inhibited both the PI3K/AKT and mitogen-activated protein kinase pathways, thereby overcoming any compensatory activation. In view of the important tumor suppressor function induced by restoring expression of microRNA (miR)-126 in metastatic melanoma cells, we examined whether miR-126 has a synergistic role when included in a triple combination alongside PIK-75 and vemurafenib. We found that enforced expression of miR-126 (which alone can reduce tumorigenicity) significantly increased PIK-75 activity when used as either a single agent or in combination with vemurafenib. Interestingly, PIK-75 proved to be effective against early passage cell lines derived from patients' biopsies and on melanoma cell lines resistant to either vemurafenib or dabrafenib, thus suggesting that it potentially has the capability to overcome drug resistance. Finally, the synergistic role played by miR-126 in combination with vemurafenib and/or PIK-75 was demonstrated in vivo in mouse xenograft models, in which tumor growth inhibition was associated with increased apoptosis. These results not only show the efficacy of PIK-75 and vemurafenib co-treatment but also indicate that restoration of miR-126 expression in advanced melanoma can enhance their antitumor activity, which may possibly allow dose reduction to decrease adverse events without reducing the therapeutic benefits.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , MAP Quinases Reguladas por Sinal Extracelular , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma , MicroRNAs/metabolismo , Proteínas de Neoplasias , Fosfatidilinositol 3-Quinases , RNA Neoplásico , Animais , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Hidrazonas/farmacologia , Sistema de Sinalização das MAP Quinases/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , MicroRNAs/genética , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Sulfonamidas/farmacologia , Vemurafenib/farmacologia
12.
Mol Cell Proteomics ; 17(5): 993-1009, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29217617

RESUMO

Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.


Assuntos
Imunoprecipitação/métodos , Complexos Multiproteicos/isolamento & purificação , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Apoptose , Biotinilação , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Técnicas de Silenciamento de Genes , Humanos , Carioferinas/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteína Fosfatase 2C/metabolismo , Proteômica , Receptores Citoplasmáticos e Nucleares/metabolismo , Reprodutibilidade dos Testes , Receptor fas/metabolismo
13.
Cell Stem Cell ; 21(1): 35-50.e9, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28602620

RESUMO

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Cadeias alfa de Integrinas/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Sistemas de Liberação de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Células HeLa , Humanos , Cadeias alfa de Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
PLoS One ; 11(5): e0156325, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27223470

RESUMO

Duchenne Muscular Dystrophy, a genetic disorder that results in a gradual breakdown of muscle, is associated to mild to severe cognitive impairment in about one-third of dystrophic patients. The brain dysfunction is independent of the muscular pathology, occurs early, and is most likely due to defects in the assembly of the Dystrophin-associated Protein Complex (DPC) during embryogenesis. We have recently described the interaction of the DPC component ß-dystrobrevin with members of complexes that regulate chromatin dynamics, and suggested that ß-dystrobrevin may play a role in the initiation of neuronal differentiation. Since oxygen concentrations and miRNAs appear as well to be involved in the cellular processes related to neuronal development, we have studied how these factors act on ß-dystrobrevin and investigated the possibility of their functional interplay using the NTera-2 cell line, a well-established model for studying neurogenesis. We followed the pattern of expression and regulation of ß-dystrobrevin during the early stages of neuronal differentiation induced by exposure to retinoic acid (RA) under hypoxia as compared with normoxia, and found that ß-dystrobrevin expression is regulated during RA-induced differentiation of NTera-2 cells. We also found that ß-dystrobrevin pattern is delayed under hypoxic conditions, together with a delay in the differentiation and an increase in the proliferation rate of cells. We identified miRNA-143 as a direct regulator of ß-dystrobrevin expression, demonstrated that ß-dystrobrevin is expressed in the nucleus and showed that, in line with our previous in vitro results, ß-dystrobrevin is a repressor of synapsin I in live cells. Altogether the newly identified regulatory pathway miR-143/ß-dystrobrevin/synapsin I provides novel insights into the functions of ß-dystrobrevin and opens up new perspectives for elucidating the molecular mechanisms underlying the neuronal involvement in muscular dystrophy.


Assuntos
Proteínas Associadas à Distrofina/genética , Proteínas Associadas à Distrofina/metabolismo , MicroRNAs/genética , Neurogênese , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Regiões 3' não Traduzidas , Diferenciação Celular , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Proliferação de Células , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Tretinoína/farmacologia
15.
Stem Cells Transl Med ; 5(4): 511-23, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26956206

RESUMO

UNLABELLED: Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]-targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)-driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC-enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC-enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti-EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance. SIGNIFICANCE: Colorectal cancer stem cells (CSCs) have been shown to be responsible for tumor propagation, metastatic dissemination, and relapse. However, molecular pathways present in CSCs, as well as mechanisms of therapy resistance, are mostly unknown. Taking advantage of genetically characterized CSC lines derived from colorectal tumors, this study provides an extensive analysis of CSC response to EGFR-targeted therapy in vivo and an overview of factors implicated in therapy response or resistance. Furthermore, the implementation of a biobank of molecularly annotated CSC lines provides an innovative resource for future investigations in colorectal cancer.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas/patologia , Animais , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Análise em Microsséries , Modelos Biológicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Transdução de Sinais/genética
16.
PLoS One ; 9(4): e94438, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24740347

RESUMO

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to exhibit therapeutic activity in cancer. However, many tumors remain resistant to treatment with TRAIL. Therefore, small molecules that potentiate the cytotoxic effects of TRAIL could be used for combinatorial therapy. Here we found that the ionophore antibiotic salinomycin acts in synergism with TRAIL, enhancing TRAIL-induced apoptosis in glioma cells. Treatment with low doses of salinomycin in combination with TRAIL augmented the activation of caspase-3 and increased TRAIL-R2 cell surface expression. TRAIL-R2 upmodulation was required for mediating the stimulatory effect of salinomycin on TRAIL-mediated apoptosis, since it was abrogated by siRNA-mediated TRAIL-R2 knockdown. Salinomycin in synergism with TRAIL exerts a marked anti-tumor effect in nude mice xenografted with human glioblastoma cells. Our results suggest that the combination of TRAIL and salinomycin may be a useful tool to overcome TRAIL resistance in glioma cells and may represent a potential drug for treatment of these tumors. Importantly, salinomycin+TRAIL were able to induce cell death of well-defined glioblastoma stem-like lines.


Assuntos
Citotoxinas/farmacologia , Glioblastoma/tratamento farmacológico , Piranos/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Camundongos Nus
17.
Cancer Cell Int ; 13(1): 101, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-24148231

RESUMO

BACKGROUND: Homeobox (HOX) genes deregulation has been largely implicated in the development of human leukemia. Among the HOXB cluster, HOXB1 was silent in a number of analyzed acute myeloid leukemia (AML) primary cells and cell lines, whereas it was expressed in normal terminally differentiated peripheral blood cells. METHODS: We evaluated the biological effects and the transcriptome changes determined by the retroviral transduction of HOXB1 in the human promyelocytic cell line HL60. RESULTS: Our results suggest that the enforced expression of HOXB1 reduces cell growth proliferation, inducing apoptosis and cell differentiation along the monocytic and granulocytic lineages. Accordingly, gene expression analysis showed the HOXB1-dependent down-regulation of some tumor promoting genes, paralleled by the up-regulation of apoptosis- and differentiation-related genes, thus supporting a tumor suppressor role for HOXB1 in AML. Finally, we indicated HOXB1 promoter hypermethylation as a mechanism responsible for HOXB1 silencing. CONCLUSIONS: We propose HOXB1 as an additional member of the HOX family with tumour suppressor properties suggesting a HOXB1/ATRA combination as a possible future therapeutic strategy in AML.

18.
Int J Cancer ; 133(4): 879-92, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23400877

RESUMO

Cutaneous melanoma is the fastest increasing cancer worldwide. Although several molecular abnormalities have been associated with melanoma progression, the underlying mechanisms are still largely unknown and few targeted therapies are under evaluation. Here we show that the HOXB7/PBX2 dimer acts as a positive transcriptional regulator of the oncogenic microRNA-221 and -222. In addition, demonstrating c-FOS as a direct target of miR-221&222, we identify a HOXB7/PBX2→miR-221&222 →c-FOS regulatory link, whereby the abrogation of functional HOXB7/PBX2 dimers leads to reduced miR-221&222 transcription and elevated c-FOS expression with consequent cell death. Taking advantage of the treatment with the peptide HXR9, an antagonist of HOX/PBX dimerization, we recognize miR-221&222 as effectors of its action, in turn confirming the HXR9 efficacy in the treatment of human melanoma malignancy, whilst sparing normal human melanocytes. Our findings, besides suggesting the potential therapeutic of HXR9 or its derivatives in malignant melanoma, suggest the disruption of the HOXB7/PBX2 complexes, miR-221&222 inhibition or even better their combination, as innovative therapeutic approaches.


Assuntos
Apoptose/fisiologia , Proteínas de Homeodomínio/genética , Melanoma/patologia , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Proteínas Proto-Oncogênicas/genética , Neoplasias Cutâneas/patologia , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Dimerização , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/fisiologia , Humanos , MicroRNAs/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , RNA Interferente Pequeno , Transcrição Genética
19.
J Cell Physiol ; 227(9): 3291-300, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22170005

RESUMO

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Colo/crescimento & desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Miofibroblastos , Adipogenia/fisiologia , Biópsia , Células da Medula Óssea/citologia , Antígeno CD146/imunologia , Antígeno CD146/metabolismo , Condrogênese/fisiologia , Colo/citologia , Hematopoese/fisiologia , Humanos , Células-Tronco Mesenquimais/citologia , Microscopia Confocal , Osteogênese/fisiologia
20.
Pigment Cell Melanoma Res ; 24(5): 953-65, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21711453

RESUMO

MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas. We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region. Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222. Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount. To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions. In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells. Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/patologia , MicroRNAs/genética , Proteína Proto-Oncogênica c-ets-1/genética , Transdução de Sinais/genética , Neoplasias Cutâneas/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética/efeitos dos fármacos
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