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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204950

RESUMO

The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, where V600EBRAF is a main oncogene. Here, we analyse the effect of V600EBRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition of V600EBRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show that V600EBRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells to V600EBRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects of V600EBRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy by V600EBRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targeting V600EBRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment of V600EBRAF-positive thyroid tumours.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Apoptose/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia
2.
Cancers (Basel) ; 13(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800291

RESUMO

Dual specificity phosphatase 1 (DUSP1) is crucial in prostate cancer (PC), since its expression is downregulated in advanced carcinomas. Here, we investigated DUSP1 effects on the expression of mesenchymal marker Snail, cell migration and invasion, analyzing the underlying mechanisms mediated by mitogen-activated protein kinases (MAPKs) inhibition. To this purpose, we used different PC cells overexpressing or lacking DUSP1 or incubated with MAPKs inhibitors. Moreover, we addressed the correlation of DUSP1 expression with Snail and activated MAPKs levels in samples from patients diagnosed with benign hyperplasia or prostate carcinoma, studying its implication in tumor prognosis and survival. We found that DUSP1 downregulates Snail expression and impairs migration and invasion in PC cells. Similar results were obtained following the inhibition of c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK). In clinical samples, we evidenced an inverse correlation between DUSP1 expression and Snail levels, which are further associated with JNK and ERK activation. Consequently, the pattern DUSP1high/activated JNKlow/activated ERKlow/Snaillow is associated with an overall extended survival of PC patients. In summary, the ratio between DUSP1 and Snail expression, with additional JNK and ERK activity measurement, may serve as a potential biomarker to predict the clinical outcome of PC patients. Furthermore, DUSP1 induction or inhibition of JNK and ERK pathways could be useful to treat PC.

3.
Leukemia ; 33(4): 981-994, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30185934

RESUMO

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin-Sca-1+c-kit+CD48-CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.


Assuntos
Aminoquinolinas/farmacologia , Autofagia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/patologia , Poliaminas/farmacologia , Animais , Apoptose , Proliferação de Células , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Células Tumorais Cultivadas
4.
FEBS J ; 286(7): 1271-1283, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30222247

RESUMO

Despite the development of selective BCR-ABL-targeting tyrosine kinase inhibitors (TKIs) transforming the management of chronic myeloid leukaemia (CML), therapy-resistant leukaemic stem cells (LSCs) persist after TKI treatment and present an obstacle to a CML cure. Recently, we and others have made significant contributions to the field by unravelling survival dependencies in LSCs to work towards the goal of eradicating LSCs in CML patients. In this review, we describe these findings focusing on autophagy and mitochondrial metabolism, which have recently been uncovered as two essential processes for LSCs quiescence and survival respectively. In addition, we discuss the therapeutic potential of autophagy and mitochondrial metabolism inhibition as a strategy to eliminate CML cells in patients where the resistance to TKI is driven by BCR-ABL-independent mechanism(s).


Assuntos
Autofagia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células-Tronco Neoplásicas/patologia , Fosforilação Oxidativa
5.
Mol Cell Oncol ; 5(1): e1403532, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29404396

RESUMO

We have recently uncovered an abnormal increase in mitochondrial oxidative metabolism in therapy-resistant chronic myeloid leukaemia stem cells (LSCs). By simultaneously disrupting mitochondrial respiration and inhibiting BCR-ABL kinase activity using the antibiotic tigecycline and imatinib respectively, we effectively eradicated LSCs and prevented disease relapse in pre-clinical animal models.

6.
J Natl Cancer Inst ; 110(5): 467-478, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29165716

RESUMO

Background: Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods: Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration-approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)-derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4-6 mice per group). All statistical tests were two-sided. Results: We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Conclusion: Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib/administração & dosagem , Imidazóis/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Terapia de Alvo Molecular/métodos , Piridazinas/administração & dosagem , Pirimidinas/administração & dosagem , Quinolinas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Med ; 23(10): 1234-1240, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28920959

RESUMO

Treatment of chronic myeloid leukemia (CML) with imatinib mesylate and other second- and/or third-generation c-Abl-specific tyrosine kinase inhibitors (TKIs) has substantially extended patient survival. However, TKIs primarily target differentiated cells and do not eliminate leukemic stem cells (LSCs). Therefore, targeting minimal residual disease to prevent acquired resistance and/or disease relapse requires identification of new LSC-selective target(s) that can be exploited therapeutically. Considering that malignant transformation involves cellular metabolic changes, which may in turn render the transformed cells susceptible to specific assaults in a selective manner, we searched for such vulnerabilities in CML LSCs. We performed metabolic analyses on both stem cell-enriched (CD34+ and CD34+CD38-) and differentiated (CD34-) cells derived from individuals with CML, and we compared the signature of these cells with that of their normal counterparts. Through combination of stable isotope-assisted metabolomics with functional assays, we demonstrate that primitive CML cells rely on upregulated oxidative metabolism for their survival. We also show that combination treatment with imatinib and tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicates CML LSCs both in vitro and in a xenotransplantation model of human CML. Our findings provide a strong rationale for investigation of the use of TKIs in combination with tigecycline to treat patients with CML with minimal residual disease.


Assuntos
Antibacterianos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Minociclina/análogos & derivados , Mitocôndrias/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Quimioterapia Combinada , Feminino , Humanos , Hipoglicemiantes/farmacologia , Mesilato de Imatinib/uso terapêutico , Técnicas In Vitro , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Espectrometria de Massas , Metabolômica , Camundongos , Camundongos Endogâmicos NOD , Minociclina/farmacologia , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fenformin/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tigeciclina , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Rev. chil. neurocir ; 42(2): 156-159, nov. 2016. ilus
Artigo em Espanhol | LILACS | ID: biblio-869769

RESUMO

El Sistema Ventricular Cerebral se desarrolla de forma paralela al resto del Sistema Nervioso Central, facilitando la circulación del Líquido Cefalorraquídeo, desde su separación del líquido amniótico a nivel embrionario. Este desarrollo es necesario para entender correctamente la anatomía ventricular y facilitar el abordaje para patologías intraventriculares. El objetivo de esta revisión es reconocer los puntos más importantes en la embriología ventricular para facilitar el aprendizaje de la anatomía quirúrgica ventricular.


The cerebral ventricular system is developed in parallel with the rest of the central nervous system, facilitating the circulation of cerebrospinal fluid, from the amniotic fluid separation in the embryonic phases. This development is necessary to correctly understand the ventricular anatomy and facilitate approach to intraventricular pathologies. The objective of this review is to recognize the most important points in the ventricular embryology and in the intraventricular endoscopic vision to facilitate learning of the ventricular surgical anatomy.


Assuntos
Humanos , Endoscopia/métodos , Ventrículos Cerebrais/embriologia , Ventriculostomia/métodos , Sistema Nervoso Central , Tubo Neural
9.
Nature ; 534(7607): 341-6, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-27281222

RESUMO

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Animais , Antígenos CD34/metabolismo , Azepinas/farmacologia , Azepinas/uso terapêutico , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Imidazolinas/farmacologia , Imidazolinas/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Camundongos , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/transplante , Proteômica , Proteínas Proto-Oncogênicas c-myc/deficiência , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Transcriptoma , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
10.
Autophagy ; 12(6): 936-48, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27168493

RESUMO

A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34(+) progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.


Assuntos
Proteína 7 Relacionada à Autofagia/metabolismo , Diferenciação Celular , Metabolismo Energético , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Cromossomo Filadélfia , Animais , Antígenos CD34/metabolismo , Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Deleção de Genes , Técnicas de Silenciamento de Genes , Glicólise/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células K562 , Análise do Fluxo Metabólico , Metaboloma/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/metabolismo
11.
Mol Carcinog ; 55(11): 1639-1654, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26392228

RESUMO

The epithelial-mesenchymal transition (EMT) is a crucial process in tumour progression, by which epithelial cells acquire a mesenchymal phenotype, increasing its motility and the ability to invade distant sites. Here, we describe the molecular mechanisms by which V600E BRAF, TGFß and the Src/FAK complex cooperatively regulate EMT induction and cell motility of anaplastic thyroid cancer cells. Analysis of EMT marker levels reveals a positive correlation between TGFß and Snail expression, with a concomitant downregulation of E-cadherin, accompanied by an increase of cell migration and invasion. Furthermore, we show that V600E BRAF depletion by siRNA or inhibition of its activity by treatment with its inhibitor PLX4720 reverses the TGFß-mediated effects on Snail, E-cadherin, migration and invasion. Moreover, V600E BRAF induces TGFß secretion through a MEK/ERK-dependent mechanism. In addition, TGFß activates the Src/FAK complex, which in turn regulates the expression of Snail and E-cadherin as well as cell migration. The inhibition of Src with the inhibitor SU6656 or abrogation of FAK expression with a specific siRNA reverses the TGFß-induced effects. Interestingly, we demonstrate that activation of the Src/FAK complex by TGFß is independent of V600E BRAF signalling, since inhibition of this oncogene does not affect its phosphorylation. Our data strongly suggest that TGFß induces EMT and aggressiveness of thyroid cancer cells by parallel mechanisms involving both the V600E BRAF/MEK/ERK and Src/FAK pathways independently. Thus, we describe novel functions for Src/FAK in mediating the EMT program and aggressiveness regulated by TGFß, establishing the inhibition of these proteins as a possible effective approach in preventing tumour progression of V600E BRAF-expressing thyroid tumours. © 2015 Wiley Periodicals, Inc.


Assuntos
Quinase 1 de Adesão Focal/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases , Mutação , Invasividade Neoplásica , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sulfonamidas/farmacologia
12.
Neoplasia ; 16(6): 529-42, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25030625

RESUMO

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma.


Assuntos
Proteínas do Olho/genética , Melanoma/genética , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fatores de Crescimento Neural/genética , Serpinas/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Senescência Celular/genética , Progressão da Doença , Epistasia Genética , Proteínas do Olho/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Metástase Neoplásica , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo
13.
Cancer Lett ; 335(1): 232-41, 2013 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-23435375

RESUMO

BRAF is a main oncogene in human thyroid cancer. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with BRAF inhibitor PLX4720 decreases migration and invasion in thyroid cancer cells expressing oncogenic (V600E)BRAF through a MEK/ERK-dependent mechanism, since treatment with the MEK inhibitor U0126 exerts the same effect. Moreover, over-expression of (V600E)BRAF increases migration and invasion of wild-type BRAF thyroid cells. Using the same strategies, we demonstrate that these effects are mediated by upregulation of the transcriptional repressor Snail with a concomitant decrease of its target E-cadherin, both hallmarks of EMT. These results reveal a novel (V600E)BRAF-induced mechanism in thyroid tumours progression and provides a rationale for using the PLX4720 inhibitor to target (V600E)BRAF signalling to effectively control progression of thyroid cancer.


Assuntos
Caderinas/metabolismo , Carcinoma/metabolismo , Indóis/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/metabolismo , Antígenos CD , Butadienos/farmacologia , Caderinas/genética , Carcinoma/patologia , Carcinoma Papilar , Linhagem Celular Tumoral , Movimento Celular , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Mutação de Sentido Incorreto , Invasividade Neoplásica , Nitrilas/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologia
14.
Cancer Lett ; 314(2): 244-55, 2012 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-22056813

RESUMO

BRAF is a main oncogene in human melanomas. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with RAF inhibitor Sorafenib induces apoptosis in NPA melanoma cells expressing oncogenic (V600E)BRAF. This effect is mediated through a MEK/ERK-independent mechanism, since treatment with the MEK inhibitor U0126 does not exert any effect. Moreover, we demonstrate that inhibition of the PI3K/AKT/mTOR cascade alone does not increase apoptosis in these cells. However, the blockage of this pathway in cells lacking either BRAF expression or activity cooperates to induce higher levels of apoptosis than those achieved by inhibition of BRAF alone. Consistently, we demonstrate that abrogation of BRAF expression increases AKT and mTOR phosphorylation, suggesting the existence of a compensatory pro-survival mechanism after BRAF depletion. Together, our data provide a rationale for dual targeting of BRAF and PI3K/AKT/mTOR signalling to effectively control melanoma disease.


Assuntos
Apoptose/efeitos dos fármacos , Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Melanoma/patologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Serina-Treonina Quinases TOR/fisiologia
15.
Carcinogenesis ; 30(10): 1670-7, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19700418

RESUMO

Cholesterol is necessary for proliferation and survival of transformed cells. Here we analyse the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in colorectal cancer cells carrying oncogenic Ras or (V600E)B-RAF mutations. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment results in a significant increase in apoptosis in HT-29 and Colo-205 cells containing the (V600E)B-RAF mutation, but not in HCT-116 and LoVo cells harbouring the (G13D)Ras mutation, or BE cells, which possess two mutations, (G13D)Ras and (G463V)B-RAF. We also demonstrate that oncogenic Ras protects from apoptosis induced by cholesterol depletion through constitutive activation of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. The specific activation of the PI3K/AKT pathway by overexpression of the (V12)RasC40 mutant or a constitutively active AKT decreases the LPDS plus 25-HC-induced apoptosis in HT-29 cells, whereas PI3K inhibition or abrogation of AKT expression renders HCT-116 sensitive to cholesterol depletion-induced apoptosis. Moreover, our data show that LPDS plus 25-HC increases the activity of c-Jun N-terminal kinase proteins only in HT-29 cells and that the inhibition of this kinase blocks the apoptosis induced by LPDS plus 25-HC. Finally, we demonstrate that AKT hyperactivation by oncogenic Ras protects from apoptosis, preventing the activation of c-Jun N-terminal kinase by cholesterol depletion. Thus, our data demonstrate that low levels of cholesterol induce apoptosis in colorectal cancer cells without oncogenic Ras mutations. These results reveal a novel molecular characteristic of colon tumours containing Ras or B-RAF mutations and should help in defining new targets for cancer therapy.


Assuntos
Apoptose/genética , Colesterol/deficiência , Genes ras/efeitos dos fármacos , Células HT29/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células 3T3 , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Colesterol/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Ativação Enzimática , Citometria de Fluxo , Genes ras/genética , Células HT29/efeitos dos fármacos , Células HT29/patologia , Humanos , Hidroxicolesteróis/farmacologia , Lipoproteínas/sangue , MAP Quinase Quinase 4/metabolismo , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Transfecção
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