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1.
Proc Natl Acad Sci U S A ; 117(16): 9064-9073, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32273388

RESUMO

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.


Assuntos
Neoplasias Encefálicas/patologia , Adesões Focais/patologia , Glioblastoma/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Patológica/patologia , Citoesqueleto de Actina/metabolismo , Animais , Encéfalo/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Estudos de Coortes , Progressão da Doença , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Intravital , Camundongos , Microscopia Confocal , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neovascularização Patológica/genética , Imagem com Lapso de Tempo , Vinculina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Proc Natl Acad Sci U S A ; 115(25): E5766-E5775, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29866840

RESUMO

The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.


Assuntos
Mama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Domínios de Homologia à Plecstrina/fisiologia , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Ciclina D1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo
3.
Br J Cancer ; 122(5): 648-657, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31857724

RESUMO

BACKGROUND: Patient-derived xenograft (PDX) mouse tumour models can predict response to therapy in patients. Predictions made from PDX cultures (PDXC) would allow for more rapid and comprehensive evaluation of potential treatment options for patients, including drug combinations. METHODS: We developed a PDX library of BRAF-mutant metastatic melanoma, and a high-throughput drug-screening (HTDS) platform utilising clinically relevant drug exposures. We then evaluated 34 antitumor agents across eight melanoma PDXCs, compared drug response to BRAF and MEK inhibitors alone or in combination with PDXC and the corresponding PDX, and investigated novel drug combinations targeting BRAF inhibitor-resistant melanoma. RESULTS: The concordance of cancer-driving mutations across patient, matched PDX and subsequent PDX generations increases as variant allele frequency (VAF) increases. There was a high correlation in the magnitude of response to BRAF and MEK inhibitors between PDXCs and corresponding PDXs. PDXCs and corresponding PDXs from metastatic melanoma patients that progressed on standard-of-care therapy demonstrated similar resistance patterns to BRAF and MEK inhibitor therapy. Importantly, HTDS identified novel drug combinations to target BRAF-resistant melanoma. CONCLUSIONS: The biological consistency observed between PDXCs and PDXs suggests that PDXCs may allow for a rapid and comprehensive identification of treatments for aggressive cancers, including combination therapies.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Melanoma/tratamento farmacológico , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Distribuição Aleatória , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Proc Natl Acad Sci U S A ; 113(22): 6254-8, 2016 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-27185926

RESUMO

Microphthalmia-associated transcription factor (MITF) plays a critical and complex role in melanocyte transformation. Although several downstream targets of MITF action have been identified, the precise mechanisms by which MITF promotes melanocytic tumor progression are incompletely understood. Recent studies identified an oncogenic role for the bromodomain plant homeodomain finger transcription factor (BPTF) gene in melanoma progression, in part through activation of BCL2, a canonical target of MITF signaling. Analysis of the BPTF promoter identified a putative MITF-binding site, suggesting that MITF may regulate BPTF expression. Overexpression of MITF resulted in up-regulation of BPTF in a panel of melanoma and melanocyte cell lines. shRNA-mediated down-regulation of MITF in melanoma cells was accompanied by down-regulation of BPTF and BPTF-regulated genes (including BCL2) and resulted in reduced proliferative capacity of melanoma cells. The suppression of cell growth mediated by MITF silencing was rescued by overexpression of BPTF cDNA. Binding of MITF to the BPTF promoter was demonstrated using ChIP analysis. MITF overexpression resulted in direct transcriptional activation of BPTF, as evidenced by increased luciferase activity driven by the BPTF promoter. These results indicate that BPTF transduces key prosurvival signals driven by MITF, further supporting its important role in promoting melanoma cell survival and progression.


Assuntos
Antígenos Nucleares/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Melanócitos/citologia , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Antígenos Nucleares/genética , Apoptose , Sítios de Ligação , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Imunofluorescência , Humanos , Luciferases/metabolismo , Melanócitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição/genética , Ativação Transcricional
5.
Mol Ther ; 23(1): 71-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25195599

RESUMO

MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3' untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Neoplasias Cutâneas/genética , Regiões 3' não Traduzidas , Animais , Apoptose , Sequência de Bases , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Proc Natl Acad Sci U S A ; 109(18): 7067-72, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22511720

RESUMO

Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma.


Assuntos
Biomarcadores Tumorais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/secundário , Melanoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Sequência de Bases , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/genética , Melanoma/secundário , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/genética , Transdução de Sinais
7.
Mol Cancer Ther ; 22(9): 1100-1111, 2023 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-37440705

RESUMO

As a result of tumor heterogeneity and solid cancers harboring multiple molecular defects, precision medicine platforms in oncology are most effective when both genetic and pharmacologic determinants of a tumor are evaluated. Expandable patient-derived xenograft (PDX) mouse tumor and corresponding PDX culture (PDXC) models recapitulate many of the biological and genetic characteristics of the original patient tumor, allowing for a comprehensive pharmacogenomic analysis. Here, the somatic mutations of 23 matched patient tumor and PDX samples encompassing four cancers were first evaluated using next-generation sequencing (NGS). 19 antitumor agents were evaluated across 78 patient-derived tumor cultures using clinically relevant drug exposures. A binarization threshold sensitivity classification determined in culture (PDXC) was used to identify tumors that best respond to drug in vivo (PDX). Using this sensitivity classification, logic models of DNA mutations were developed for 19 antitumor agents to predict drug response. We determined that the concordance of somatic mutations across patient and corresponding PDX samples increased as variant allele frequency increased. Notable individual PDXC responses to specific drugs, as well as lineage-specific drug responses were identified. Robust responses identified in PDXC were recapitulated in vivo in PDX-bearing mice and logic modeling determined somatic gene mutation(s) defining response to specific antitumor agents. In conclusion, combining NGS of primary patient tumors, high-throughput drug screen using clinically relevant doses, and logic modeling, can provide a platform for understanding response to therapeutic drugs targeting cancer.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Animais , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Testes Farmacogenômicos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Antineoplásicos/farmacologia , Mutação
8.
J Biol Chem ; 286(19): 16606-14, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21454583

RESUMO

MicroRNAs (miRNAs) regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-205 is significantly suppressed in melanoma specimens when compared with nevi and is correlated inversely with melanoma progression. miRNA target databases predicted E2F1 and E2F5 as putative targets. The expression levels of E2F1 and E2F5 were correlated inversely with that of miR-205 in melanoma cell lines. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequences complementary to either E2F1 or E2F5. Overexpression of miR-205 in melanoma cells reduced E2F1 and E2F5 protein levels. The proliferative capacity of melanoma cells was suppressed by miR-205 and mediated by E2F-regulated AKT phosphorylation. miR-205 overexpression resulted in induction of apoptosis, as evidenced by increased cleaved caspase-3, poly-(ADP-ribose) polymerase, and cytochrome c release. Stable overexpression of miR-205 suppressed melanoma cell proliferation, colony formation, and tumor cell growth in vivo and induced a senescence phenotype accompanied by elevated expression of p16INK4A and other markers for senescence. E2F1 overexpression in miR-205-expressing cells partially reversed the effects on melanoma cell growth and senescence. These results demonstrate a novel role for miR-205 as a tumor suppressor in melanoma.


Assuntos
Fator de Transcrição E2F1/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , MicroRNAs/metabolismo , Neoplasias Cutâneas/metabolismo , Regiões 3' não Traduzidas , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Senescência Celular , Fator de Transcrição E2F5/metabolismo , Humanos , Modelos Biológicos , Poli(ADP-Ribose) Polimerases/metabolismo
9.
J Pers Med ; 12(11)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36579573

RESUMO

We describe our institutional experience of developing a liquid biopsy approach using circulating tumor DNA (ctDNA) analysis for personalized medicine in cancer patients, focusing on the hurdles encountered during the multistep process in order to benefit other investigators wishing to set up this type of study in their institution. Blood samples were collected at the time of cancer surgery from 209 patients with one of nine different cancer types. Extracted tumor DNA and circulating cell-free DNA were sequenced using cancer-specific panels and the Illumina MiSeq machine. Almost half of the pairs investigated were uninformative, mostly because there was no trackable pathogenic mutation detected in the original tumor. The pairs with interpretable data corresponded to 107 patients. Analysis of 48 gene sequences common to both panels was performed and revealed that about 40% of these pairs contained at least one driver mutation detected in the DNA extracted from plasma. Here, we describe the choice of our overall approach, the selection of the cancer panels, and the difficulties encountered during the multistep process, including the use of several tumor types and in the data analysis. We also describe some case reports using longitudinal samples, illustrating the potential advantages and rewards in performing ctDNA sequencing to monitor tumor burden or guide treatment for cancer patients.

10.
Cancer Res ; 81(11): 2956-2969, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33766890

RESUMO

Melanoma occurs as a consequence of inherited susceptibility to the disease and exposure to UV radiation (UVR) and is characterized by uncontrolled cellular proliferation and a high mutational load. The precise mechanisms by which UVR contributes to the development of melanoma remain poorly understood. Here we show that activation of nuclear receptor coactivator 3 (NCOA3) promotes melanomagenesis through regulation of UVR sensitivity, cell-cycle progression, and circumvention of the DNA damage response (DDR). Downregulation of NCOA3 expression, either by genetic silencing or small-molecule inhibition, significantly suppressed melanoma proliferation in melanoma cell lines and patient-derived xenografts. NCOA3 silencing suppressed expression of xeroderma pigmentosum C and increased melanoma cell sensitivity to UVR. Suppression of NCOA3 expression led to activation of DDR effectors and reduced expression of cyclin B1, resulting in G2-M arrest and mitotic catastrophe. A SNP in NCOA3 (T960T) reduced NCOA3 protein expression and was associated with decreased melanoma risk, given a significantly lower prevalence in a familial melanoma cohort than in a control cohort without cancer. Overexpression of wild-type NCOA3 promoted melanocyte survival following UVR and was accompanied by increased levels of UVR-induced DNA damage, both of which were attenuated by overexpression of NCOA3 (T960T). These results describe NCOA3-regulated pathways by which melanoma can develop, with germline NCOA3 polymorphisms enabling enhanced melanocyte survival in the setting of UVR exposure, despite an increased mutational burden. They also identify NCOA3 as a novel therapeutic target for melanoma. SIGNIFICANCE: This study explores NCOA3 as a regulator of the DDR and a therapeutic target in melanoma, where activation of NCOA3 contributes to melanoma development following exposure to ultraviolet light.


Assuntos
Biomarcadores Tumorais/metabolismo , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Melanoma/patologia , Coativador 3 de Receptor Nuclear/metabolismo , Lesões por Radiação/patologia , Raios Ultravioleta/efeitos adversos , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Humanos , Melanoma/etiologia , Melanoma/metabolismo , Camundongos , Camundongos Nus , Mutação , Coativador 3 de Receptor Nuclear/genética , Lesões por Radiação/etiologia , Lesões por Radiação/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
J Invest Dermatol ; 141(8): 2028-2036.e2, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33610559

RESUMO

Homologous recombination DNA damage repair (HR-DDR) deficient patients with various solid tumors have been treated with PARP inhibitors. However, the clinical characteristics of patients with melanoma who have HR-DDR gene mutations and the consequences of PARP inhibition are poorly understood. We compared the commercially available next-generation sequencing data from 84 patients with melanomas from our institution with a dataset of 1,986 patients as well as 1,088 patients profiled in cBioportal. In total, 21.4% of patients had ≥1 functional HR-DDR mutation, most commonly involving BRCA1, ARID1A, ATM, ATR, and FANCA. Concurrent NF1, BRAF, and NRAS mutations were found in 39%, 39%, and 22% of cases, respectively. HR-DDR gene mutation was associated with high tumor mutational burden and clinical response to checkpoint blockade. A higher prevalence of HR-DDR mutations was observed in the datasets from Foundation Medicine (Cambridge, CA) and those from the Cancer Genome Atlas. Treatment of HR-DDR‒mutated patient-derived xenograft models of melanoma with PARP inhibitor produced significant antitumor activity in vivo and was associated with increased apoptotic activity. RNA sequencing analysis of PARP inhibitor-treated tumors indicated alterations in the pathways involving extracellular matrix remodeling, cell adhesion, and cell-cycle progression. Melanomas with HR-DDR mutations represent a unique subset, which is more likely to benefit from checkpoint blockade and may be targeted with PARP inhibitor.


Assuntos
Biomarcadores Tumorais/genética , Melanoma/genética , Reparo de DNA por Recombinação/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Dano ao DNA/efeitos dos fármacos , Análise Mutacional de DNA/estatística & dados numéricos , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Masculino , Melanoma/tratamento farmacológico , Melanoma/epidemiologia , Camundongos , Pessoa de Meia-Idade , Epidemiologia Molecular , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Prevalência , Intervalo Livre de Progressão , RNA-Seq , Reparo de DNA por Recombinação/efeitos dos fármacos , Estudos Retrospectivos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/epidemiologia , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
12.
BMC Biotechnol ; 10: 9, 2010 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-20144189

RESUMO

BACKGROUND: The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. RESULTS: Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. CONCLUSIONS: In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.


Assuntos
DNA/química , Transfecção/métodos , Animais , Linhagem Celular , Sobrevivência Celular , Eletroporação , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Lipídeos/química , Camundongos , Polietilenoimina/química , Coelhos , Suínos
13.
Am J Pathol ; 174(3): 1009-16, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19179607

RESUMO

IkappaBgamma is one member of a family of proteins that can inhibit the nuclear localization of nuclear factor-kappaB. However, the other specific functions of IkappaBgamma are still poorly understood, and its effects on tumor metastasis have not yet been characterized. We examined the consequences of targeting IkappaBgamma in melanoma cells using a hammerhead ribozyme. We developed stable transformant B16-F10 melanoma cell lines that express a ribozyme that targets mouse IkappaBgamma (IkappaBgamma-144-Rz). Tail-vein injection of B16-F10 cells that stably express IkappaBgamma-144-Rz into mice resulted in a significant reduction of the metastatic potential of these cells. IkappaBgamma-144-Rz-expressing B16 cells were shown to have increased transcriptional activity of nuclear factor-kappaB. We then showed that IkappaBgamma-144-Rz-expressing cells demonstrated both reduced invasion and increased apoptosis, suggesting the existence of pathways through which IkappaBgamma promotes melanoma metastasis. Using gene expression profiling, we identified a differentially expressed gene set that is regulated by the stable suppression of IkappaBgamma that may participate in mediating its anti-metastatic effects; we also confirmed the altered expression levels of several of these genes by quantitative real time polymerase chain reaction. Plasmid-mediated expression of IkappaBgamma-144-Rz produced a significant inhibition of the metastatic progression of B16-F10 cells to the lung and resulted in significant anti-invasive and pro-apoptotic effects on murine Lewis lung carcinoma cells. Our results suggest a novel role for IkappaBgamma in promoting the metastatic progression of melanoma.


Assuntos
Melanoma Experimental/genética , Melanoma Experimental/patologia , Subunidade p50 de NF-kappa B/genética , NF-kappa B/genética , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/prevenção & controle , RNA Catalítico/genética , Animais , Clonagem Molecular , Citometria de Fluxo , Camundongos , NF-kappa B/antagonistas & inibidores , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , Transcrição Gênica , Células Tumorais Cultivadas
14.
Sci Rep ; 10(1): 18489, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116269

RESUMO

Cholangiocarcinoma (CCA) is a highly invasive cancer, diagnosed at an advanced stage, and refractory to surgical intervention and chemotherapy. Cyclin-dependent kinases (CDKs) regulate cell cycle progression and transcriptional processes, and are considered potential therapeutic targets for cancer. Dinaciclib is a small molecule multi-CDK inhibitor targeting CDK 2/5/9. In this study, the therapeutic efficacy of dinaciclib was assessed using patient-derived xenograft cells (PDXC) and CCA cell lines. Treatment with dinaciclib significantly suppressed cell proliferation, induced caspase 3/7 levels and apoptotic activity in PDXC and CCA cell lines. Dinaciclib suppressed expression of its molecular targets CDK2/5/9, and anti-apoptotic BCL-XL and BCL2 proteins. Despite the presence of cyclin D1 amplification in the PDXC line, palbociclib treatment had no effect on cell proliferation, cell cycle or apoptosis in the PDXC as well as other CCA cell lines. Importantly, dinaciclib, in combination with gemcitabine, produced a robust and sustained inhibition of tumor progression in vivo in a PDX mouse model, greater than either of the treatments alone. Expression levels of two proliferative markers, phospho-histone H3 and Ki-67, were substantially suppressed in samples treated with the combination regimen. Our results identify dinaciclib as a novel and potent therapeutic agent alone or in combination with gemcitabine for the treatment of CCA.


Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Óxidos N-Cíclicos/farmacologia , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Neoplasias Gastrointestinais/tratamento farmacológico , Indolizinas/farmacologia , Compostos de Piridínio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Antígeno Ki-67 , Masculino , Camundongos , Camundongos Endogâmicos NOD , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/metabolismo , Gencitabina
15.
Oncogenesis ; 8(8): 42, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409772

RESUMO

Cholangiocarcinoma (CCA) is a rare, highly invasive malignancy, and its incidence is increasing globally. MicroRNAs (miRNAs) mediate a wide array of cellular and biological processes and are dysregulated in various tumors. The functional and biological roles of miRNAs in CCA have not been fully elucidated. In this study, we show that miR-876 expression levels and copy number are significantly attenuated in the TCGA cohort of CCA tissue samples. TCGA expression data was consistent with the observed substantial decrease in miR-876 expression in patient samples and CCA cell lines. In-silico algorithm databases revealed BCL-XL as a potential target of miR-876. We observed miR-876 expression to be downregulated, whereas, BCL-XL upregulated in CCA cell lines. BCL-XL was identified as a direct functional target of miR-876 in CCA. miR-876-mediated reduction of BCL-XL regulated cell survival, induced apoptosis and caspase 3/7 expression in CCA. BCL-XL overexpression reversed the miR-876 mediated effect on CCA cell growth and apoptosis. Stable overexpression of miR-876 produced potent tumor suppressor activity and in vivo tumor cell growth reduction. Overexpression of miR-876 in a patient-derived xenograft (PDX) cell line significantly suppressed BCL-XL expression and spheroid formation with a concomitant induction of caspase 3/7 activity and apoptosis. This study demonstrates a novel tumor suppressor role for miR-876 in CCA, identifies BCL-XL as an actionable target, and suggests a potential therapeutic role for miR-876 in CCA.

16.
Clin Cancer Res ; 24(17): 4119-4125, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29776954

RESUMO

Purpose: Previous studies have indicated an important role for pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis. Here we aimed to confirm the role of PHIP copy number in successive stages of melanoma progression.Experimental Design:PHIP copy number was examined using FISH in three independent cohorts by recording the percentage of cells harboring ≥3 copies of PHIP The impact of PHIP copy number on survival was assessed using Cox regression analysis. The enrichment of PHIP was assessed in various molecular melanoma subtypes. PHIP expression was analyzed in The Cancer Genome Atlas (TCGA) melanoma cohort.Results: Elevated PHIP copy number was significantly predictive of reduced distant metastasis-free survival (DMFS) and disease-specific survival (DSS), and increased prevalence of ulceration in primary melanoma (cohort No. 1). By multivariate analysis, PHIP FISH scores were independently predictive of DMFS and DSS. PHIP copy number was enriched in metastatic melanomas harboring mutant NRAS or expressing PTEN protein (cohort No. 2). PHIP copy number was significantly elevated in metastatic melanomas when compared with matched primary tumors from the same patient (cohort No. 3). Several of these associations were replicated using TCGA cohort analysis.Conclusions: These results underscore the important role of PHIP copy-number elevation in melanoma progression, and identify molecular subtypes of melanoma in which PHIP is enriched. Finally, as elevated PHIP copy number appears to be selected for during the progression of primary to metastatic melanoma, these results confirm PHIP as a promising therapeutic target for melanoma. Clin Cancer Res; 24(17); 4119-25. ©2018 AACR.


Assuntos
Biomarcadores Tumorais/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/genética , Prognóstico , Neoplasias Cutâneas/genética , Idoso , Variações do Número de Cópias de DNA/genética , Progressão da Doença , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Cutâneas/patologia , Melanoma Maligno Cutâneo
17.
Curr Gene Ther ; 6(4): 481-504, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16918336

RESUMO

As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cell's endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.


Assuntos
Reparo do DNA/fisiologia , Marcação de Genes , Terapia Genética , Animais , Humanos
18.
Oligonucleotides ; 16(4): 375-86, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17155912

RESUMO

Oligonucleotides can mediate sequence-specific gene modification that results in the correction and/or alteration of genomic DNA. There is evidence to suggest that the polymerase chain reaction (PCR)-based analytical methods usually used to analyze oligonucleotide-mediated modification can generate artifacts. To investigate the conditions under which a PCR artifact can be generated and eliminated when analyzing small fragment homologous replacement (SHFR)-mediated modification, cells homozygous for the DeltaF508 mutation (CFBE41o-) were mixed with small DNA fragments (SDFs) containing the wild-type CFTR (wt-CFTR) sequence. An artifact could be generated after wild-type allele-specific PCR (wtAS-PCR) if the genomic DNA was not gel purified. Without gel purification, the amount of SDF/cell required to generate the artifact was dependent to the AS primer pairs used. When the genomic DNA was gel purified, no artifact could be detected with any of the wtAS-PCR primers whether the SDF was mixed with the cells or transfected into the cells. Furthermore, treatment of cellular mRNA with DNase was sufficient to eliminate potential artifacts in the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, it is critical to gel purify genomic DNA and DNase treat mRNA when analyzing SFHR-mediated modification by PCR.


Assuntos
DNA/genética , DNA/isolamento & purificação , Sequência de Bases , Linhagem Celular , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , DNA/química , Eletroforese em Gel de Ágar , Conversão Gênica , Humanos , Mutação , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , Transfecção
19.
Oncotarget ; 7(15): 19519-30, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-26799586

RESUMO

UNLABELLED: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis, which lacks effective targeted therapies. There is an urgent need to better understand the underlying molecular mechanisms of TNBC aggressiveness and identify novel, efficient targets for therapeutic intervention. METHODS: miRNA qRT-PCR was used to determine the expression of miR-1296 in cell lines. The miR-1296 overexpression effects in TNBC cell lines were investigated using assays of colony formation, cell cycle and apoptosis. Immunoblotting was performed to determine the expression of the miR-1296 target protein, and luciferase assays were performed to confirm the target of miR-1296 action. RESULTS: miR-1296 expression was significantly suppressed in TNBC cell lines and tissues samples. Overexpression of miR-1296 significantly suppressed cell proliferation of two TNBC cell lines when compared to control miRNA-expressing cells. A significant decrease in the S-phase of the cell cycle was observed following miR-1296 overexpression, accompanied by induction of apoptosis in TNBC cells. Cyclin D1 (CCND1) was identified as a target of miR-1296 action. miR-1296 overexpression significantly suppressed the luciferase activity of reporter plasmid containing the 3'UTR of CCND1 and protein expression levels of CCND1 in TNBC cells. The effects of miR-1296 overexpression on TNBC cell growth were reversed by CCND1 overexpression. miR-1296 expression sensitized TNBC cells to cisplatin treatment. CONCLUSION: Our results demonstrate a novel tumor suppressor role for miR-1296 in triple-negative breast cancer cell lines, identify CCND1 as its target of action, and demonstrate a potential role for miR-1296 in sensitizing breast cancer cells to cisplatin.


Assuntos
Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , MicroRNAs/genética , Neoplasias de Mama Triplo Negativas/genética , Regiões 3' não Traduzidas/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Ciclina D1/metabolismo , Humanos , Immunoblotting , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
20.
J Natl Cancer Inst ; 107(5)2015 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-25713167

RESUMO

BACKGROUND: Bromodomain PHD finger transcription factor (BPTF) plays an important role in chromatin remodeling, but its functional role in tumor progression is incompletely understood. Here we explore the oncogenic effects of BPTF in melanoma. METHODS: The consequences of differential expression of BPTF were explored using shRNA-mediated knockdown in several melanoma cell lines. Immunoblotting was used to assess the expression of various proteins regulated by BPTF. The functional role of BPTF in melanoma progression was investigated using assays of colony formation, invasion, cell cycle, sensitivity to selective BRAF inhibitors, and in xenograft models of melanoma progression (n = 12 mice per group). The biomarker role of BPTF in melanoma progression was assessed using fluorescence in situ hybridization and immunohistochemical analyses. All statistical tests were two-sided. RESULTS: shRNA-mediated BPTF silencing suppressed the proliferative capacity (by 65.5%) and metastatic potential (by 66.4%) of melanoma cells. Elevated BPTF copy number (mean ≥ 3) was observed in 28 of 77 (36.4%) melanomas. BPTF overexpression predicted poor survival in a cohort of 311 melanoma patients (distant metastasis-free survival P = .03, and disease-specific survival P = .008), and promoted resistance to BRAF inhibitors in melanoma cell lines. Metastatic melanoma tumors progressing on BRAF inhibitors contained low BPTF-expressing, apoptotic tumor cell subclones, indicating the continued presence of drug-responsive subclones within tumors demonstrating overall resistance to anti-BRAF agents. CONCLUSIONS: These studies demonstrate multiple protumorigenic functions for BPTF and identify it as a novel target for anticancer therapy. They also suggest the combination of BPTF targeting with BRAF inhibitors as a novel therapeutic strategy for melanomas with mutant BRAF.


Assuntos
Antígenos Nucleares/metabolismo , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Imidazóis/farmacologia , Indóis/farmacologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Terapia de Alvo Molecular , Proteínas do Tecido Nervoso/metabolismo , Oximas/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Sulfonamidas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Progressão da Doença , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Terapia de Alvo Molecular/métodos , Mutação/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Cutâneas/genética , Vemurafenib , Ensaios Antitumorais Modelo de Xenoenxerto
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