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1.
Cell ; 185(12): 2071-2085.e12, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35561684

RESUMO

Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.


Assuntos
Melanoma , Nevo Pigmentado , Neoplasias Cutâneas , Animais , Xenoenxertos , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Transplante de Neoplasias , Nevo Pigmentado/congênito , Nevo Pigmentado/tratamento farmacológico , Nevo Pigmentado/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controle
2.
Proc Natl Acad Sci U S A ; 119(30): e2119048119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35858411

RESUMO

The major challenges in pancreatic ductal adenocarcinoma (PDAC) management are local or distant metastasis and limited targeted therapeutics to prevent it. To identify a druggable target in tumor secretome and to explore its therapeutic intervention, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of tumors obtained from a patient-derived xenograft model of PDAC. Galectin-3 binding protein (Gal-3BP) is identified as a highly secreted protein, and its overexpression is further validated in multiple PDAC tumors and primary cells. Knockdown and exogenous treatment of Gal-3BP showed that it is required for PDAC cell proliferation, migration, and invasion. Mechanistically, we revealed that Gal-3BP enhances galectin-3-mediated epidermal growth factor receptor signaling, leading to increased cMyc and epithelial-mesenchymal transition. To explore the clinical impact of these findings, two antibody clones were developed, and they profoundly abrogated the metastasis of PDAC cells in vivo. Altogether, our data demonstrate that Gal-3BP is an important therapeutic target in PDAC, and we propose its blockade by antibody as a therapeutic option for suppressing PDAC metastasis.


Assuntos
Antígenos de Neoplasias , Antineoplásicos Imunológicos , Biomarcadores Tumorais , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cromatografia Líquida , Transição Epitelial-Mesenquimal , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Proteômica , Secretoma , Espectrometria de Massas em Tandem , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Development ; 139(8): 1405-16, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22434867

RESUMO

MicroRNAs (miRNAs) regulate the expression of many mammalian genes and play key roles in embryonic hair follicle development; however, little is known of their functions in postnatal hair growth. We compared the effects of deleting the essential miRNA biogenesis enzymes Drosha and Dicer in mouse skin epithelial cells at successive postnatal time points. Deletion of either Drosha or Dicer during an established growth phase (anagen) caused failure of hair follicles to enter a normal catagen regression phase, eventual follicular degradation and stem cell loss. Deletion of Drosha or Dicer in resting phase follicles did not affect follicular structure or epithelial stem cell maintenance, and stimulation of anagen by hair plucking caused follicular proliferation and formation of a primitive transient amplifying matrix population. However, mutant matrix cells exhibited apoptosis and DNA damage and hair follicles rapidly degraded. Hair follicle defects at early time points post-deletion occurred in the absence of inflammation, but a dermal inflammatory response and hyperproliferation of interfollicular epidermis accompanied subsequent hair follicle degradation. These data reveal multiple functions for Drosha and Dicer in suppressing DNA damage in rapidly proliferating follicular matrix cells, facilitating catagen and maintaining follicular structures and their associated stem cells. Although Drosha and Dicer each possess independent non-miRNA-related functions, the similarity in phenotypes of the inducible epidermal Drosha and Dicer mutants indicates that these defects result primarily from failure of miRNA processing. Consistent with this, Dicer deletion resulted in the upregulation of multiple direct targets of the highly expressed epithelial miRNA miR-205.


Assuntos
RNA Helicases DEAD-box/genética , Deleção de Genes , MicroRNAs/metabolismo , Ribonuclease III/genética , Pele/crescimento & desenvolvimento , Animais , Cruzamentos Genéticos , RNA Helicases DEAD-box/fisiologia , Células Epidérmicas , Folículo Piloso/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Fenótipo , Ribonuclease III/fisiologia , Transdução de Sinais , Pele/metabolismo , Células-Tronco/citologia , Cicatrização
4.
Anal Chem ; 86(1): 535-42, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24199994

RESUMO

Contemporary cancer therapy refers to treatment based on genetic abnormalities found in patient's tumor. However, this approach is faced with numerous challenges, including tumor heterogeneity and molecular evolution, insufficient tumor samples available along with genetic information linking to clinical outcomes, lack of therapeutic drugs containing pharmacogenomic information, and technical limitations of rapid drug efficacy tests with insufficient quantities of primary cancer cells from patients. To address these problems and improve clinical outcomes of current personalized gene-targeted cancer therapy, we have developed a micropillar/microwell chip platform, which is ideally suited for encapsulating primary cancer cells in nanoscale spots of hydrogels on the chip, generating efficacy data with various drugs, eventually allowing for a comparison of the in vitro data obtained from the chip with clinical data as well as gene expression data. As a proof of concept in this study, we have encapsulated a U251 brain cancer cell line and three primary brain cancer cells from patients (448T, 464T, and 775T) in 30 nL droplets of alginate and then tested the therapeutic efficacy of 24 anticancer drugs by measuring their dose responses. As a result, the IC50 values of 24 anticancer drugs obtained from the brain cancer cells clearly showed patient cell-specific efficacy, some of which were well-correlated with their oncogene overexpression (c-Met and FGFR1) as well as the in vivo previous results of the mouse xenograft model with the three primary brain cancer cells.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Ensaios de Triagem em Larga Escala/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Antineoplásicos/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Resultado do Tratamento , Células Tumorais Cultivadas
5.
Small ; 10(24): 5098-105, 2014 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-25227876

RESUMO

The limiting dilution assay (LDA) is a clonogenic drug efficacy test designed to determine a value for drug efficacy based on an all-or-none (positive or negative) response within replicates. It also attempts to calculate minimum cell numbers for cells to form colony in each drugged conditions, wherein a large value implies high drug efficacy (as a large number of extant cells are required to start a colony). However, traditional LDAs are time-consuming to set up, often requiring many replicates for statistical analysis, and manual colony identification under a microscope to determine a positive or negative response is tedious and is susceptible to human error. To address these issues, a high-throughput miniaturized LDA assay is developed using a micropillar/microwell chip platform using an automatic colony identification method. Three glioblastoma multiforme (GBM) brain tumor isolates (448T, 464T, and 775T) are used to test this new assay, using the c-Met kinase inhibitors SU11274 and PHA665752 as the target drugs. The results show that the minimum cell number of 775T is larger than that of the other two cell types (SU11274 and PHA665752) in both the sampled drugs, a result that is in good agreement with the results of previous conventional experiments using 96 well plates.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Miniaturização , Ensaios de Triagem em Larga Escala , Humanos
6.
Thyroid ; 34(9): 1150-1162, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38666696

RESUMO

Background: The density of tumor-associated macrophages in the tumor microenvironment of anaplastic thyroid cancer (ATC) is associated with poor prognosis. However, the crosstalk between macrophages and ATC cells is poorly understood. This study aimed to examine the impact of macrophages on cancer cell phenotypes. We found a new mediator between M2 macrophages and ATC cells through proteomics analysis. Methods: The role of macrophages in proliferation, migration, and invasion of ATC cells was evaluated using coculture assay and conditioned medium (CM). Secretory factors in the CM from single or coculture were identified using liquid chromatography-tandem mass spectrometry proteomics analysis. We evaluated the role of the secretory factor in proliferation, migration, and invasion of cancer cells. In vivo xenograft model was used to evaluate the effect of the factor. Results: M2 macrophages significantly increased the proliferation, migration, and invasion of ATC cells, whereas M1 macrophages decreased the proliferation, migration, and invasion of ATC cells. Based on proteomic analysis of CM, we identify carboxypeptidase A4 (CPA4) as a mediator of the crosstalk between macrophages and ATC cells. CPA4 was only detected in the coculture media of M2 macrophage/8505C, and its expression in cancer cells increased by M2 macrophage. The expression of CPA4 protein was significantly higher in human thyroid cancers, particularly in ATCs, than normal and benign tissues. A bioinformatics analysis of public data revealed that CPA4 expression was associated with poor prognosis and dedifferentiation of thyroid cancer. Knockdown of CPA4 suppressed proliferation, colony formation, migration, and invasion of ATC cells, consistent with the decrease of STAT3, ERK, and AKT/mTOR phosphorylation and epithelial-mesenchymal transition (EMT) marker expression. In addition, the increased expression of CPA4 in cancer cells by M2 macrophage stimulation induced the polarization of macrophages to the M2 phenotype, which formed a positive feedback loop. Xenograft tumors did not develop after CPA4 knockdown. Conclusions: Our data suggest that CPA4 stimulates the progression of thyroid cancer by mediating between M2 macrophages and ATC cells. CPA4 can be a new therapeutic target for the treatment of patients with ATC.


Assuntos
Carboxipeptidases A , Movimento Celular , Proliferação de Células , Progressão da Doença , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Carcinoma Anaplásico da Tireoide/patologia , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Linhagem Celular Tumoral , Animais , Carboxipeptidases A/metabolismo , Carboxipeptidases A/genética , Microambiente Tumoral , Camundongos , Macrófagos Associados a Tumor/metabolismo , Macrófagos/metabolismo , Técnicas de Cocultura , Invasividade Neoplásica , Proteômica
7.
Lab Invest ; 93(3): 344-53, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23381625

RESUMO

Radiotherapy is the most widely used therapeutic modality in brain metastasis; however, it only provides palliation due to inevitable tumor recurrence. Resistance of tumor cells to ionizing radiation is a major cause of treatment failure. A critical unmet need in oncology is to develop rationale driven approaches that can enhance the efficacy of radiotherapy against metastatic tumor. Utilizing in vivo orthotopic primary tumor and brain metastasis models that recapitulate clinical situation of the patients with metastatic breast cancer, we investigated a molecular mechanism through which metastatic tumor cells acquire resistance to radiation. Recent studies have demonstrated that the hepatocyte growth factor (HGF)-c-Met pathway is essential for the pathologic development and progression of many human cancers such as proliferation, invasion and resistance to anticancer therapies. In this study, c-Met signaling activity as well as total c-Met expression was significantly upregulated in both breast cancer cell lines irradiated in vitro and ex vivo radio-resistant cells derived from breast cancer brain metastatic xenografts. To interrogate the role of c-Met signaling in radioresistance of brain metastasis, we evaluated the effects on tumor cell viability, clonogenicity, sensitivity to radiation, and in vitro/in vivo tumor growth after targeting c-Met by small-hairpin RNA (shRNA) or small-molecule kinase inhibitor (PF-2341066). Although c-Met silencing or radiation alone demonstrated a modest decrease in clonogenic growth of parental breast cancers and brain metastatic derivatives, combination of two modalities showed synergistic antitumor effects resulting in significant prolongation of overall survival in tumor-bearing mice. Taken together, optimizing c-Met targeting in combination with radiation is critical to enhance the effectiveness of radiotherapy in the treatments of brain metastasis.


Assuntos
Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Quimiorradioterapia/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Análise de Variância , Animais , Crizotinibe , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Immunoblotting , Camundongos , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirazóis , Piridinas/farmacologia , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos da radiação , Células Tumorais Cultivadas
8.
Biochem Biophys Res Commun ; 421(2): 305-11, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22708120

RESUMO

Interleukin (IL)-33, an IL-1 family member, acts as an extracellular cytokine by binding its cognate receptor, ST2. IL-33 is also a chromatin-binding transcriptional regulator highly expressed in the nuclei of endothelial cells. However, the function of IL-33 as a nuclear factor is poorly defined. Here, we show that IL-33 is a novel transcriptional regulator of the p65 subunit of the NF-κB complex and is involved in endothelial cell activation. Quantitative reverse transcriptase PCR and Western blot analyses indicated that IL-33 mediates the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in endothelial cells basally and in response to tumor necrosis factor-a-treatment. IL-33-induced ICAM-1/VCAM-1 expression was dependent on the regulatory effect of IL-33 on the nuclear factor (NF)-κB pathway; NF-κB p65 expression was enhanced by IL-33 overexpression and, conversely, reduced by IL-33 knockdown. Moreover, NF-κB p65 promoter activity and chromatin immunoprecipitation analysis revealed that IL-33 binds to the p65 promoter region in the nucleus. Our data provide the first evidence that IL-33 in the nucleus of endothelial cells participates in inflammatory reactions as a transcriptional regulator of NF-κB p65.


Assuntos
Núcleo Celular/metabolismo , Células Endoteliais/metabolismo , Interleucinas/metabolismo , Fator de Transcrição RelA/genética , Ativação Transcricional , Adesão Celular/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/genética , Molécula 1 de Adesão Intercelular/genética , Interleucina-33 , Monócitos/fisiologia , Regiões Promotoras Genéticas , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética
9.
Circ Res ; 107(7): 871-6, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20671239

RESUMO

RATIONALE: Although recent studies have suggested a role for the receptor activator of nuclear factor κB ligand (RANKL) in the late stages of atherosclerosis (eg, plaque destabilization and rupture), the underlying mechanisms and subsequent events are unclear. OBJECTIVE: Because blood clotting is common after plaque rupture, we hypothesized that RANKL influenced tissue factor (TF) expression and activity to initiate the coagulation cascade. METHODS AND RESULTS: RANKL increased the TF mRNA level and procoagulant activity in macrophages, as determined by semiquantitative reverse transcription polymerase chain reaction (semiquantitative RT-PCR) and a chromogenic assay. TF promoter analysis revealed that AP-1 and Egr-1 are responsible for RANKL-induced TF transcription. In addition, RANKL increased phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2. RANKL-induced TF expression was attenuated by JNK- and MEK1-specific inhibitors and by small interfering RNA knockdown of c-Jun and Egr-1. CONCLUSION: Our results indicate that RANKL induces TF in macrophages mainly through the cooperative action of AP-1 and Egr-1 via JNK and ERK1/2 pathways. These findings provide strong mechanistic support for the role of RANKL in the thrombogenicity of atherosclerotic plaques.


Assuntos
Aterosclerose/fisiopatologia , Macrófagos Peritoneais/fisiologia , Ligante RANK/genética , Tromboplastina/genética , Trombose/fisiopatologia , Animais , Aterosclerose/epidemiologia , Aterosclerose/metabolismo , Linhagem Celular , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Fosforilação/fisiologia , Ligante RANK/metabolismo , Ligante RANK/farmacologia , RNA Mensageiro , RNA Interferente Pequeno , Fatores de Risco , Tromboplastina/metabolismo , Trombose/epidemiologia , Trombose/metabolismo , Transcrição Gênica/fisiologia , Regulação para Cima/fisiologia
10.
Endocr Relat Cancer ; 29(6): 307-319, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35343921

RESUMO

Dabrafenib is a BRAF kinase inhibitor approved for treatment of BRAF-mutated anaplastic thyroid carcinoma (ATC) in combination with trametinib. Erlotinib is a tyrosine kinase inhibitor of EGF receptor (EGFR). We evaluated effects of dabrafenib and erlotinib combination treatment on ATC cells in vitro and in vivo. Cell proliferation, colony formation, apoptosis, and migration of ATC cells harboring a BRAF mutation (BHT101, 8505C, and SW1736) were evaluated after treatment with dabrafenib in combination with erlotinib or trametinib. The changes in activation of mitogen extracellular kinase (MEK) and extracellular signal-related kinase (ERK) signaling were also evaluated by Western blot analysis. Effects of these combinations were also evaluated using an in vivo xenograft model. First, we detected EGFR activation in dabrafenib-resistant SW1736 cells using a phospho-receptor tyrosine kinase array. A dabrafenib and erlotinib combination synergistically inhibited cell proliferation, colony formation, and migration, with an induction of apoptotic cell death in all three ATC cells, compared with dabrafenib or erlotinib alone. This synergistic effect was comparable with a dabrafenib and trametinib combination. The dabrafenib and erlotinib combination effectively inhibited phosphorylated (p)-MEK, p-ERK, and p-EGFR expressions compared with dabrafenib or erlotinib alone, while the dabrafenib and trametinib combination only inhibited p-MEK and p-ERK expressions. The dabrafenib with erlotinib or trametinib combinations also significantly suppressed tumor growth and induced apoptosis in a BHT101 xenograft model. The dabrafenib and erlotinib combination could be a potential novel treatment regimen to overcome drug resistance to dabrafenib alone in patients with BRAF-mutated ATC.


Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Receptores ErbB , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Humanos , Imidazóis , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Oximas , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética
11.
Blood ; 114(14): 3117-26, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19661270

RESUMO

Interleukin-33 (IL-33), a member of the IL-1 cytokine family, is emerging as a new regulator of immune responses and inflammatory vascular diseases. Although IL-33 and its cognate receptor ST2 appear to be expressed in vascular cells, the precise role of IL-33 in the vasculature has not been determined. In this study, we report a novel role of IL-33 as a potent endothelial activator, promoting both angiogenesis and vascular permeability. IL-33 increased proliferation, migration, and morphologic differentiation of human endothelial cells, consistently with increased angiogenesis in vivo. IL-33 also increased endothelial permeability with reduced vascular endothelial-cadherin-facilitated cell-cell junctions in vitro and induced vascular leakage in mouse skin. These effects of IL-33 were blocked by knockdown of ST2. Ligation of IL-33 with ST2 rapidly increased endothelial nitric oxide (NO) production through TRAF6-mediated activation of phosphoinoside-3-kinase, Akt, and endothelial NO synthase. Moreover, pharmacologic or genetic blockage of endothelial NO generation resulted in the inhibition of angiogenesis and vascular hyperpermeability induced by IL-33. These data demonstrate that IL-33 promotes angiogenesis and vascular leakage by stimulating endothelial NO production via the ST2/TRAF6-Akt-eNOS signaling pathway. These findings open new perspectives for the role of IL-33 in the pathogenesis of angiogenesis-dependent and inflammatory vascular diseases.


Assuntos
Interleucinas/metabolismo , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Receptores de Superfície Celular/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Western Blotting , Permeabilidade Capilar , Movimento Celular , Proliferação de Células , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Imunofluorescência , Humanos , Imunoprecipitação , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/genética
12.
Dev Biol ; 329(2): 227-41, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19269284

RESUMO

The transcription factor Elf5 plays an important role in mammary gland development. However, because of the embryonic lethality of Elf5 straight knockout mice, prior studies have been limited to experiments with Elf5 haploinsufficient animals, overexpression systems or transplants. Here, we have utilized K14-Cre to generate mammary-gland specific Elf5 conditional knockout mice. During pregnancy, Elf5-null mammary epithelium completely failed to initiate alveologenesis, and a characteristic of virgin ductal epithelial cells persisted postpartum. We demonstrate that the loss of Elf5 leads to the absence of alveolar secretory markers confirming previous published data. Interestingly, the developmental block due to a lack of Elf5 could not be restored by multiple gestations. Elf5-null mammary epithelial cells also display disorganized cell structures as evident by altered cell polarities, which might be the cause for collapsed lumina. We observe reduced levels of Stat5 and attenuated Stat5 activity as measured by p-Stat5 levels both in Elf5-null mammary glands as well as cultured mammary epithelial cells. This data suggests that the failure of alveolar and lactogenic differentiation due to the loss of Elf5 is mediated in part due to impaired Stat5 activity. In support of this hypothesis, we show by ChIP experiments that Stat5a promoter contains a conserved Elf5-binding site that is occupied by Elf5 in mammary glands. Mammary epithelia lacking Elf5 exhibited downregulation of several other critical genes involved in alveologenesis, suggesting Elf5 as a master regulator in alveolar development. We propose a model for Elf5-mediated alveolar development, in which Elf5 regulates the expression of key mediators of the PrlR/Jak2/Stat5 signaling pathway.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Glândulas Mamárias Animais/embriologia , Fator de Transcrição STAT5/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/genética , Regulação para Baixo/fisiologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia
13.
Cancer Lett ; 465: 82-93, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31404615

RESUMO

Pancreatic cancer is one of the leading causes of cancer death, mainly due to the absence of early diagnostic tool and effective therapeutic agents. To identify an effective therapeutic agent for pancreatic ductal adenocarcinoma cells (PDAC), we used 10 Gene Expression Omnibus (GEO) data sets and L1000CDS2 pharmacogenetic search tool and obtained chemical "perturvants" that were predicted to reverse the abnormal gene expression changes in PDAC. Among 20 initial candidates, we measured IC50 for six compounds and identified BX-795, PDK1/TBK1 inhibitor, as a therapeutic candidate. We found that BX-795 inhibits primary PDAC cell proliferation more effectively than normal cells. Following molecular analysis revealed that BX-795 down-regulates mTOR-GSK3ß pathway and trigger apoptosis. Moreover, we found that BX-795 suppresses primary PDAC cell migration via downregulation of Snail and Slug. Finally, efficacy test in patient-derived xenograft model of PDAC showed BX-795 can inhibit in vivo tumor growth as efficient as gemcitabine and a combination with trametinib further suppresses tumor growth. Collectively, these results demonstrate the BX-795 as an effective therapeutic candidate for PDAC treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Piridonas/administração & dosagem , Pirimidinas/administração & dosagem , Pirimidinonas/administração & dosagem , Tiofenos/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Neoplasias Pancreáticas/genética , Testes Farmacogenômicos , Piridonas/farmacologia , Pirimidinas/farmacologia , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Tiofenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Mol Oncol ; 12(9): 1526-1539, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30055072

RESUMO

Pancreatic cancer is one of the most difficult cancers to cure due to the lack of early diagnostic tools and effective therapeutic agents. In this study, we aimed to isolate new bioactive compounds that effectively kill pancreatic ductal adenocarcinoma (PDAC) cells, but not untransformed, human pancreatic ductal epithelial (HPDE) cells. To this end, we established four primary PDAC cell lines and screened 4141 compounds from four bioactive-compound libraries. Initial screening yielded 113 primary hit compounds that caused over a 50% viability reduction in all tested PDAC cells. Subsequent triplicate, dose-dependent analysis revealed three compounds with a tumor cell-specific cytotoxic effect. We found that these three compounds fall into a single category of thiopurine biogenesis. Among them, 6-thioguanine (6-TG) showed an IC50 of 0.39-1.13 µm toward PDAC cells but had no effect on HPDE cells. We propose that this cancer selectivity is due to differences in thiopurine methyltransferase (TPMT) expression between normal and cancer cells. This enzyme is responsible for methylation of thiopurine, which reduces its cytotoxicity. We found that TPMT levels were lower in all four PDAC cell lines than in HPDE or Panc1 cells, and that knockdown of TPMT in HPDE or Panc1 cells sensitized them to 6-TG. Lastly, we used a patient-derived xenograft model to confirm that 6-TG has a significant antitumor effect in combination with gemcitabine. Overall, our study presents 6-TG as a strong candidate for use as a therapeutic agent against PDAC with low levels of TPMT.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Tioguanina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/enzimologia , Linhagem Celular Tumoral , Desoxicitidina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metiltransferases/biossíntese , Neoplasias Pancreáticas/enzimologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
15.
Biochem J ; 398(3): 497-507, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16704374

RESUMO

The ESE (epithelium-specific Ets) subfamily of Ets transcription factors plays an important role in regulating gene expression in a variety of epithelial cell types. Although ESE proteins have been shown to bind to regulatory elements of some epithelial genes, the optimal DNA-binding sequence has not been experimentally ascertained for any member of the ESE subfamily of transcription factors. This has made the identification and validation of their targets difficult. We are studying ESE-2 (Elf5), which is highly expressed in epithelial cells of many tissues including skin keratinocytes. Here, we identify the preferred DNA-binding site of ESE-2 by performing CASTing (cyclic amplification and selection of targets) experiments. Our analysis shows that the optimal ESE-2 consensus motif consists of a GGA core and an AT-rich 5'- and 3'-flanking sequences. Mutational and competition experiments demonstrate that the flanking sequences that confer high DNA-binding affinity for ESE-2 show considerable differences from the known consensus DNA-binding sites of other Ets proteins, thus reinforcing the idea that the flanking sequences may impart recognition specificity for Ets proteins. In addition, we have identified a novel isoform of murine ESE-2, ESE-2L, that is generated by use of a hitherto unreported new exon and an alternate promoter. Interestingly, transient transfection assays with an optimal ESE-2 responsive reporter show that both ESE-2 and ESE-2L are weak transactivators. However, similar studies utilizing GAL4 chimaeras of ESE-2 demonstrate that while the DNA-binding ETS (E twenty-six) domain functions as a repressor, the PNT (pointed domain) of ESE-2 can act as a potent transcriptional activation domain. This novel transactivating property of PNT is also shared by ESE-3, another ESE family member. Identification of the ESE-2 consensus site and characterization of the transcriptional activation properties of ESE-2 shed new light on its potential as a regulator of target genes.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA , Humanos , Camundongos , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ets , Fatores de Transcrição/genética , Ativação Transcricional
16.
Oncotarget ; 7(38): 62533-62546, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27613834

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is the most challenging type of cancer to treat, with a 5-year survival rate of <10%. Furthermore, because of the large portion of the inoperable cases, it is difficult to obtain specimens to study the biology of the tumors. Therefore, a patient-derived xenograft (PDX) model is an attractive option for preserving and expanding these tumors for translational research. Here we report the generation and characterization of 20 PDX models of PDAC. The success rate of the initial graft was 74% and most tumors were re-transplantable. Histological analysis of the PDXs and primary tumors revealed a conserved expression pattern of p53 and SMAD4; an exome single nucleotide polymorphism (SNP) array and Comprehensive Cancer Panel showed that PDXs retained over 94% of cancer-associated variants. In addition, Polyphen2 and the Sorting Intolerant from Tolerant (SIFT) prediction identified 623 variants among the functional SNPs, highlighting the heterologous nature of pancreatic PDXs; an analysis of 409 tumor suppressor genes and oncogenes in Comprehensive Cancer Panel revealed heterologous cancer gene mutation profiles for each PDX-primary tumor pair. Altogether, we expect these PDX models are a promising platform for screening novel therapeutic agents and diagnostic markers for the detection and eradication of PDAC.


Assuntos
Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Polimorfismo de Nucleotídeo Único , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Exoma , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Variação Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise Multivariada , Mutação , Neoplasias Pancreáticas/patologia , República da Coreia , Transdução de Sinais , Proteína Smad4/genética , Proteína Supressora de Tumor p53/genética , Neoplasias Pancreáticas
17.
Oncotarget ; 6(24): 20370-87, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26284487

RESUMO

The roles of oncogenic miRNAs are widely recognized in many cancers. Inhibition of single miRNA using antagomiR can efficiently knock-down a specific miRNA. However, the effect is transient and often results in subtle phenotype, as there are other miRNAs contribute to tumorigenesis. Here we report a multi-potent miRNA sponge inhibiting multiple miRNAs simultaneously. As a model system, we targeted miR-21, miR-155 and miR-221/222, known as oncogenic miRNAs in multiple tumors including breast and pancreatic cancers. To achieve efficient knockdown, we generated perfect and bulged-matched miRNA binding sites (MBS) and introduced multiple copies of MBS, ranging from one to five, in the multi-potent miRNA sponge. Luciferase reporter assay showed the multi-potent miRNA sponge efficiently inhibited 4 miRNAs in breast and pancreatic cancer cells. Furthermore, a stable and inducible version of the multi-potent miRNA sponge cell line showed the miRNA sponge efficiently reduces the level of 4 target miRNAs and increase target protein level of these oncogenic miRNAs. Finally, we showed the miRNA sponge sensitize cells to cancer drug and attenuate cell migratory activity. Altogether, our study demonstrates the multi-potent miRNA sponge is a useful tool to examine the functional impact of simultaneous inhibition of multiple miRNAs and proposes a therapeutic potential.


Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Poríferos/genética , Poríferos/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Transfecção
18.
PLoS One ; 10(10): e0137703, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26444992

RESUMO

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM.


Assuntos
Neoplasias Encefálicas/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Fatores de Transcrição Forkhead/genética , Glioblastoma/patologia , Fatores de Transcrição SOXB1/genética , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/terapia , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Glioblastoma/mortalidade , Glioblastoma/terapia , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Células-Tronco Neoplásicas/citologia , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno , Tolerância a Radiação/genética , Fatores de Transcrição SOXB1/biossíntese , Transdução de Sinais/genética
19.
Cell Res ; 24(10): 1157-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25033756

RESUMO

Ultraviolet radiation (UVR) is a major risk factor for melanoma development, but it has been unclear exactly how UVR leads to melanomagenesis. In a recent publication in Nature, Viros et al. identify TP53/Trp53 as a UVR-target gene in melanoma and show that UVR-induced TP53/Trp53 mutations accelerate BRAF(V600E)-driven melanomagenesis.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/efeitos da radiação , Melanoma/genética , Melanoma/patologia , Mutagênese/efeitos da radiação , Proteínas Proto-Oncogênicas B-raf/genética , Proteína Supressora de Tumor p53/genética , Raios Ultravioleta/efeitos adversos , Animais , Feminino , Humanos
20.
PLoS One ; 9(8): e103327, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25084005

RESUMO

Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient's surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.


Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Medicina de Precisão , Pesquisa Translacional Biomédica , Adulto , Idoso , Animais , Antineoplásicos/farmacologia , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/mortalidade , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genômica , Glioblastoma/diagnóstico , Glioblastoma/tratamento farmacológico , Glioblastoma/mortalidade , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Farmacogenética , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
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