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1.
Cell Rep ; 37(3): 109870, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34686346

RESUMO

FBXO31 is the substrate receptor of one of many CUL1-RING ubiquitin ligase (CRL1) complexes. Here, we show that low FBXO31 mRNA levels are associated with high pre-operative prostate-specific antigen (PSA) levels and Gleason grade in human prostate cancer. Mechanistically, the ubiquitin ligase CRL1FBXO31 promotes the ubiquitylation-mediated degradation of DUSP6, a dual specificity phosphatase that dephosphorylates and inactivates the extracellular-signal-regulated kinase-1 and -2 (ERK1/2). Depletion of FBXO31 stabilizes DUSP6, suppresses ERK signaling, and activates the PI3K-AKT signaling cascade. Moreover, deletion of FBXO31 promotes tumor development in a mouse orthotopic model of prostate cancer. Treatment with BCI, a small molecule inhibitor of DUSP6, suppresses AKT activation and prevents tumor formation, suggesting that the FBXO31 tumor suppressor activity is dependent on DUSP6. Taken together, our studies highlight the relevance of the FBXO31-DUSP6 axis in the regulation of ERK- and PI3K-AKT-mediated signaling pathways, as well as its therapeutic potential in prostate cancer.


Assuntos
Fosfatase 6 de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas F-Box/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas Culina/genética , Proteínas Culina/metabolismo , Cicloexilaminas/farmacologia , Fosfatase 6 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 6 de Especificidade Dupla/genética , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Proteínas F-Box/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Indenos/farmacologia , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteólise , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Int J Mol Sci ; 22(18)2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34575852

RESUMO

Pancreatic cancer is an aggressive disease with poor prognosis. Only about 15-20% of patients diagnosed with pancreatic cancer can undergo surgical resection, while the remaining 80% are diagnosed with locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). In these cases, chemotherapy and radiotherapy only confer marginal survival benefit. Recent progress has been made in understanding the pathobiology of pancreatic cancer, with a particular effort in discovering new diagnostic and prognostic biomarkers, novel therapeutic targets, and biomarkers that can predict response to chemo- and/or radiotherapy. Mitochondria have become a focus in pancreatic cancer research due to their roles as powerhouses of the cell, important subcellular biosynthetic factories, and crucial determinants of cell survival and response to chemotherapy. Changes in the mitochondrial genome (mtDNA) have been implicated in chemoresistance and metastatic progression in some cancer types. There is also growing evidence that changes in microRNAs that regulate the expression of mtDNA-encoded mitochondrial proteins (mitomiRs) or nuclear-encoded mitochondrial proteins (mitochondria-related miRs) could serve as diagnostic and prognostic cancer biomarkers. This review discusses the current knowledge on the clinical significance of changes of mtDNA, mitomiRs, and mitochondria-related miRs in pancreatic cancer and their potential role as predictors of cancer risk, as diagnostic and prognostic biomarkers, and as molecular targets for personalized cancer therapy.


Assuntos
Biomarcadores Tumorais , Genes Mitocondriais , MicroRNAs/genética , Neoplasias Pancreáticas/genética , DNA Mitocondrial , Regulação Neoplásica da Expressão Gênica , Variação Genética , Haplótipos , Humanos , Mitocôndrias/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/mortalidade , Polimorfismo de Nucleotídeo Único , Prognóstico
4.
Cells ; 10(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34359935

RESUMO

Programmed cell death is a conserved evolutionary process of cell suicide that is central to the development and integrity of eukaryotic organisms [...].


Assuntos
Apoptose , Doença , Saúde , Animais , Apoptose/efeitos dos fármacos , Produtos Biológicos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caspase 2/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/patologia , Degeneração Neural/patologia
5.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361078

RESUMO

Mitochondria are complex intracellular organelles traditionally identified as the powerhouses of eukaryotic cells due to their central role in bioenergetic metabolism. In recent decades, the growing interest in mitochondria research has revealed that these multifunctional organelles are more than just the cell powerhouses, playing many other key roles as signaling platforms that regulate cell metabolism, proliferation, death and immunological response. As key regulators, mitochondria, when dysfunctional, are involved in the pathogenesis of a wide range of metabolic, neurodegenerative, immune and neoplastic disorders. Far more recently, mitochondria attracted renewed attention from the scientific community for their ability of intercellular translocation that can involve whole mitochondria, mitochondrial genome or other mitochondrial components. The intercellular transport of mitochondria, defined as horizontal mitochondrial transfer, can occur in mammalian cells both in vitro and in vivo, and in physiological and pathological conditions. Mitochondrial transfer can provide an exogenous mitochondrial source, replenishing dysfunctional mitochondria, thereby improving mitochondrial faults or, as in in the case of tumor cells, changing their functional skills and response to chemotherapy. In this review, we will provide an overview of the state of the art of the up-to-date knowledge on intercellular trafficking of mitochondria by discussing its biological relevance, mode and mechanisms underlying the process and its involvement in different pathophysiological contexts, highlighting its therapeutic potential for diseases with mitochondrial dysfunction primarily involved in their pathogenesis.


Assuntos
Doenças Metabólicas/fisiopatologia , Mitocôndrias/fisiologia , Dinâmica Mitocondrial , Neoplasias/fisiopatologia , Doenças Neurodegenerativas/fisiopatologia , Animais , Humanos , Doenças Metabólicas/terapia , Neoplasias/terapia
6.
Cells ; 10(6)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205944

RESUMO

Pancreatic cancer (PC) is among the most devastating digestive tract cancers worldwide. This cancer is characterized by poor diagnostic detection, lack of therapy, and difficulty in predicting tumorigenesis progression. Although mutations of key oncogenes and oncosuppressor involved in tumor growth and in immunosurveillance escape are known, the underlying mechanisms that orchestrate PC initiation and progression are poorly understood or still under debate. In recent years, the attention of many researchers has been concentrated on the role of extracellular vesicles and of a particular subset of extracellular vesicles, known as exosomes. Literature data report that these nanovesicles are able to deliver their cargos to recipient cells playing key roles in the pathogenesis and progression of many pancreatic precancerous conditions. In this review, we have summarized and discussed principal cargos of extracellular vesicles characterized in PC, such as miRNAs, lncRNAs, and several proteins, to offer a systematic overview of their function in PC progression. The study of extracellular vesicles is allowing to understand that investigation of their secretion and analysis of their content might represent a new and potential diagnostic and prognostic tools for PC.


Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Animais , Vesículas Extracelulares/patologia , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Prognóstico
7.
Int J Mol Sci ; 22(4)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671549

RESUMO

Tumor cell invasion depends largely on degradation of the extracellular matrix (ECM) by protease-rich structures called invadopodia, whose formation and activity requires the convergence of signaling pathways engaged in cell adhesion, actin assembly, membrane regulation and ECM proteolysis. It is known that ß1-integrin stimulates invadopodia function through an invadopodial p(T567)-ezrin/NHERF1/NHE1 signal complex that regulates NHE1-driven invadopodia proteolytic activity and invasion. However, the link between ß1-integrin and this signaling complex is unknown. In this study, in metastatic breast (MDA-MB-231) and prostate (PC-3) cancer cells, we report that integrin-linked kinase (ILK) integrates ß1-integrin with this signaling complex to regulate invadopodia activity and invasion. Proximity ligation assay experiments demonstrate that, in invadopodia, ILK associates with ß1-integrin, NHE1 and the scaffold proteins p(T567)-ezrin and NHERF1. Activation of ß1-integrin increased both invasion and invadopodia activity, which were specifically blocked by inhibition of either NHE1 or ILK. We conclude that ILK integrates ß1-integrin with the ECM proteolytic/invasion signal module to induce NHE1-driven invadopodial ECM proteolysis and cell invasion.


Assuntos
Integrina beta1/metabolismo , Podossomos/metabolismo , Podossomos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Humanos , Masculino , Células PC-3 , Fosfoproteínas/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
8.
Biology (Basel) ; 9(12)2020 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-33353059

RESUMO

Mitochondria are highly dynamic and regulated organelles implicated in a variety of important functions in the cell, including energy production, fatty acid metabolism, iron homeostasis, programmed cell death, and cell signaling. Changes in mitochondrial metabolism, signaling and dynamics are hallmarks of cancer. Understanding whether these modifications are associated with alterations of the mitochondrial proteome is particularly relevant from a translational point of view because it may contribute to better understanding the molecular bases of cancer development and progression and may provide new potential prognostic and diagnostic biomarkers as well as novel molecular targets for anti-cancer treatment. Making an inventory of the mitochondrial proteins has been particularly challenging given that there is no unique consensus targeting sequence that directs protein import into mitochondria, some proteins are present at very low levels, while other proteins are expressed only in some cell types, in a particular developmental stage or under specific stress conditions. This review aims at providing the state-of-the-art on methodologies used to characterize the mitochondrial proteome in tumors and highlighting the biological relevance of changes in expression and delocalization of proteins in and out the mitochondria in cancer biology.

9.
Mol Cell Oncol ; 7(6): 1833698, 2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33235922

RESUMO

Epigenetic reprogramming is emerging as a key mechanism for metastasis development. Our study identified a novel regulatory mechanism whereby promoter methylation-mediated epigenetic silencing of the gene encoding the ubiquitin ligase subunit F-box/LRR-repeat protein 7 (FBXL7) induces accumulation of active c-SRC, which, in turn, activates epithelial-to-mesenchymal transition and supports cancer cell invasion and metastasis.

10.
Nat Cell Biol ; 22(9): 1130-1142, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32839549

RESUMO

Epigenetic plasticity is a pivotal factor that drives metastasis. Here, we show that the promoter of the gene that encodes the ubiquitin ligase subunit FBXL7 is hypermethylated in advanced prostate and pancreatic cancers, correlating with decreased FBXL7 mRNA and protein levels. Low FBXL7 mRNA levels are predictive of poor survival in patients with pancreatic and prostatic cancers. FBXL7 mediates the ubiquitylation and proteasomal degradation of active c-SRC after its phosphorylation at Ser 104. The DNA-demethylating agent decitabine recovers FBXL7 expression and limits epithelial-to-mesenchymal transition and cell invasion in a c-SRC-dependent manner. In vivo, FBXL7-depleted cancer cells form tumours with a high metastatic burden. Silencing of c-SRC or treatment with the c-SRC inhibitor dasatinib together with FBXL7 depletion prevents metastases. Furthermore, decitabine reduces metastases derived from prostate and pancreatic cancer cells in a FBXL7-dependent manner. Collectively, this research implicates FBXL7 as a metastasis-suppressor gene and suggests therapeutic strategies to counteract metastatic dissemination of pancreatic and prostatic cancer cells.


Assuntos
Epigênese Genética/genética , Transição Epitelial-Mesenquimal/genética , Proteínas F-Box/genética , Inativação Gênica/fisiologia , Metástase Neoplásica/genética , Subunidades Proteicas/genética , Quinases da Família src/genética , Animais , Linhagem Celular , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células PC-3 , Transdução de Sinais/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
11.
J Clin Med ; 9(6)2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32599695

RESUMO

Mitochondria play a crucial role in cell life and death by regulating bioenergetic and biosynthetic pathways. They are able to adapt rapidly to different microenvironmental stressors by accommodating the metabolic and biosynthetic needs of the cell. Mounting evidence places mitochondrial dysfunction at the core of several diseases, notably in the context of pathologies of the cardiovascular and central nervous system. In addition, mutations in some mitochondrial proteins are bona fide cancer drivers. Better understanding of the functions of these multifaceted organelles and their components may finetune our knowledge on the molecular bases of certain diseases and suggest new therapeutic avenues.

12.
J Clin Med ; 8(11)2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31731601

RESUMO

Aging is a major risk factor for developing cancer, suggesting that these two events may represent two sides of the same coin. It is becoming clear that some mechanisms involved in the aging process are shared with tumorigenesis, through convergent or divergent pathways. Increasing evidence supports a role for mitochondrial dysfunction in promoting aging and in supporting tumorigenesis and cancer progression to a metastatic phenotype. Here, a summary of the current knowledge of three aspects of mitochondrial biology that link mitochondria to aging and cancer is presented. In particular, the focus is on mutations and changes in content of the mitochondrial genome, activation of mitochondria-to-nucleus signaling and the newly discovered mitochondria-telomere communication.

13.
Cancers (Basel) ; 11(7)2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31284411

RESUMO

Background: Mutations in the oncosuppressor gene BReast CAncer susceptibility gene 2 (BRCA2) predispose to aggressive forms of prostate cancer which show poor response to taxane-based therapy, the standard treatment for castration-resistant, aggressive prostate cancer. Herein, we addressed the question whether changes in BRCA2 expression, a potential surrogate marker for BRCA2 activity, may affect the response of castration-resistant prostate cancer cells to 6-thioguanine (6-TG), a thiopurine used in the treatment of haematological malignancies. Methods: Yeast, normal prostate cells and castration-resistant prostate cancer cells were treated with 6-TG or its analogues, in presence or absence of paclitaxel, or with olaparib, a poly-(ADP-ribose) polymerase (PARP) inhibitor currently in clinical trials for treatment of metastatic castration-resistant prostate cancer, and cell proliferation, apoptosis and androgen receptor (AR) levels were measured. Results: 6-TG inhibited cell proliferation in yeast, normal and castration-resistant prostate cancer cells but promoted apoptosis only in cancer cells. Suppression of BRCA2 expression by siRNA or shRNA increased the sensitivity to 6-TG- and olaparib-induced apoptosis but did not affect cancer cell response to taxane. Intriguingly, 6-TG reduced AR expression levels independently on BRCA2 expression. Instead, olaparib decreased AR levels only in BRCA2-knockdown prostate cancer cells. Notably, overexpression of BRCA2 resulted in resistance of castration-resistant prostate cancer cells to 6-TG-, taxane- and olaparib-based treatment but promoted sensitivity to apoptosis induced by 2-amino-6-bromopurine and 2,6-dithiopurine, two 6-TG analogues. Conclusions: Our results provide a pre-clinical rationale for the use of 6-TG in the treatment of BRCA2-deficient castration-resistant prostate cancers, and of certain 6-TG analogues for treatment of BRCA2-proficient prostate cancers.

14.
Cancer Res ; 79(6): 1098-1112, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30504123

RESUMO

IFNγ, a potent cytokine known to modulate tumor immunity and tumoricidal effects, is highly elevated in patients with prostate cancer after radiation. In this study, we demonstrate that IFNγ can induce epithelial-to-mesenchymal transition (EMT) in prostate cancer cells via the JAK-STAT signaling pathway, leading to the transcription of IFN-stimulated genes (ISG) such as IFN-induced tetratricopeptide repeat 5 (IFIT5). We unveil a new function of IFIT5 complex in degrading precursor miRNAs (pre-miRNA) that includes pre-miR-363 from the miR-106a-363 cluster as well as pre-miR-101 and pre-miR-128, who share a similar 5'-end structure with pre-miR-363. These suppressive miRNAs exerted a similar function by targeting EMT transcription factors in prostate cancer cells. Depletion of IFIT5 decreased IFNγ-induced cell invasiveness in vitro and lung metastasis in vivo. IFIT5 was highly elevated in high-grade prostate cancer and its expression inversely correlated with these suppressive miRNAs. Altogether, this study unveils a prometastatic role of the IFNγ pathway via a new mechanism of action, which raises concerns about its clinical application.Significance: A unique IFIT5-XRN1 complex involved in the turnover of specific tumor suppressive microRNAs is the underlying mechanism of IFNγ-induced epithelial-to-mesenchymal transition in prostate cancer.See related commentary by Liu and Gao, p. 1032.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Neoplasias Pulmonares/secundário , MicroRNAs/genética , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/patologia , Animais , Antivirais/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos SCID , Proteínas de Neoplasias/genética , Prognóstico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Front Oncol ; 7: 295, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29250487

RESUMO

Epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells to assume mesenchymal features, endowing them with enhanced motility and invasiveness, thus enabling cancer dissemination and metastatic spread. The induction of EMT is orchestrated by EMT-inducing transcription factors that switch on the expression of "mesenchymal" genes and switch off the expression of "epithelial" genes. Mitochondrial dysfunction is a hallmark of cancer and has been associated with progression to a metastatic and drug-resistant phenotype. The mechanistic link between metastasis and mitochondrial dysfunction is gradually emerging. The discovery that mitochondrial dysfunction owing to deregulated mitophagy, depletion of the mitochondrial genome (mitochondrial DNA) or mutations in Krebs' cycle enzymes, such as succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase, activate the EMT gene signature has provided evidence that mitochondrial dysfunction and EMT are interconnected. In this review, we provide an overview of the current knowledge on the role of different types of mitochondrial dysfunction in inducing EMT in cancer cells. We place emphasis on recent advances in the identification of signaling components in the mito-nuclear communication network initiated by dysfunctional mitochondria that promote cellular remodeling and EMT activation in cancer cells.

16.
Biochim Biophys Acta Bioenerg ; 1858(8): 686-699, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28161329

RESUMO

Mitochondria, known for more than a century as the energy powerhouse of a cell, represent key intracellular signaling hub that are emerging as important determinants of several aspects of cancer development and progression, including metabolic reprogramming, acquisition of metastatic capability, and response to chemotherapeutic drugs. The majority of cancer cells harbors somatic mutations in the mitochondrial genome (mtDNA) and/or alterations in the mtDNA content, leading to mitochondrial dysfunction. Decreased mtDNA content is also detected in tumor-initiating cells, a subpopulation of cancer cells that are believed to play an integral role in cancer recurrence following chemotherapy. Although mutations in mitochondrial genes are common in cancer cells, they do not shut down completely the mitochondrial energy metabolism and functionality. Instead, they promote rewiring of the bioenergetics and biosynthetic profile of a cancer cell through a mitochondria-to-nucleus signaling activated by "dysfunctional" mitochondria that results in changes in transcription and/or activity of cancer-related genes and signaling pathways. Different cancer cell types may undergo different bioenergetic changes, some to more glycolytic and some to more oxidative. These different metabolic signatures may coexist within the same tumor mass (intra-tumor heterogeneity). In this review we describe the current understanding of mitochondrial dysfunction in the context of cancer chemoresistance with special attention to the role of mtDNA alterations. We put emphasis on potential therapeutic strategies targeting different metabolic events specific to cancer cells, including glycolysis, glutaminolysis, oxidative phosphorylation, and the retrograde signaling, to prevent chemoresistance. We also highlight novel genome-editing strategies aimed at "correcting" mtDNA defects in cancer cells. We conclude on the importance of considering intratumor metabolic heterogeneity to develop effective metabolism-based cancer therapy that can overcome chemoresistance. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Transformação Celular Neoplásica , DNA Mitocondrial/genética , Progressão da Doença , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transferência Genética Horizontal , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Modelos Biológicos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fosforilação Oxidativa , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos
17.
Int J Oncol ; 48(6): 2722, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27082245

RESUMO

After the publication of the article the authors noted the following errors in the assembling of the figures. In Fig. 3A the tubulin panel for PC-3 cells is incorrect. The correct panel is reported below. In Figs. 3B and 5B the panels are incorrect. The correct panels are shown below. These changes do not affect the interpretation of the data or conclusions of this work [the original article was published in the International Journal of Oncology 30: 217-224, 2007; DOI: 10.3892/ijo.30.1.217].

18.
J Vis Exp ; (102): e52849, 2015 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-26327352

RESUMO

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 °C instead of the conventional 70-100 °C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other 'large' proteins like BRCA2 should be readily achievable.


Assuntos
Proteína BRCA2/genética , Inativação Gênica , Genes BRCA2 , Proteína BRCA2/biossíntese , Proteína BRCA2/deficiência , Linhagem Celular , Células Epiteliais , Técnicas de Silenciamento de Genes/métodos , Humanos , Immunoblotting/métodos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção
19.
Oncotarget ; 5(15): 6425-36, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25115390

RESUMO

Altered DAB2IP gene expression often detected in prostate cancer (PCa) is due to epigenetic silencing. In this study, we unveil a new mechanism leading to the loss of DAB2IP protein; an oncogenic S-phase kinase-associated protein-2 (Skp2) as E3 ubiquitin ligase plays a key regulator in DAB2IP degradation. In order to unveil the role of Skp2 in the turnover of DAB2IP protein, both prostate cell lines and prostate cancer specimens with a variety of molecular and cell biologic techniques were employed. We demonstrated that DAB2IP is regulated by Skp2-mediated proteasome degradation in the prostate cell lines. Further analyses identified the N-terminal DAB2IP containing the ubiquitination site. Immunohistochemical study exhibited an inverse correlation between DAB2IP and Skp2 protein expression in the prostate cancer tissue microarray. In contrast, DAB2IP can suppressSkp2 protein expression is mediated through Akt signaling. The reciprocal regulation between DAB2IP and Skp2 can impact on the growth of PCa cells. This reciprocal regulation between DAB2IP and Skp2 protein represents a unique homeostatic balance between tumor suppressor and oncoprotein in normal prostate epithelia, which is apparently altered in cancer cells. The outcome of this study has identified new potential targets for developing new therapeutic strategy for PCa.


Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Expressão Gênica , Células HEK293 , Homeostase , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/genética , Proteínas Quinases Associadas a Fase S/genética , Transfecção , Ubiquitinação , Proteínas Ativadoras de ras GTPase/genética
20.
Biochem Pharmacol ; 92(1): 62-72, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25107705

RESUMO

Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome can contribute to development of chemoresistance in malignant tumors. In this review we will describe the current knowledge on the role of mitochondrial dysfunction in the development of chemoresistance in cancer. We will also discuss the significance of this research topic in the context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, with a particular focus on the potential use of PARP inhibitors in cancer patients displaying mitochondrial DNA mutations. We will discuss recent studies highlighting the importance of the cross-talk between the tumor microenvironment and mitochondrial functionality in determining selective response to certain chemotherapeutic drugs. Finally, owing to the similarities between cancer and yeast cell metabolism, we will point out the use of yeast as a model system to study cancer-related genes and for anti-cancer drugs screening.


Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mitocôndrias/patologia , Mutação
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