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1.
Sci Adv ; 9(19): eade0059, 2023 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-37172086

RESUMEN

CRISPR-Cas9 has been used successfully to introduce indels in somatic cells of rodents; however, precise editing of single nucleotides has been hampered by limitations of flexibility and efficiency. Here, we report technological modifications to the CRISPR-Cas9 vector system that now allows homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic tissues to generate tumor models with high flexibility and efficiency. Somatic editing of either Kras or Pik3ca in both normal and hyperplastic mammary glands led to swift tumorigenesis. The resulting tumors shared some histological, transcriptome, and proteome features with tumors induced by lentivirus-mediated expression of the respective oncogenes, but they also exhibited some distinct characteristics, particularly showing less intertumor variation, thus potentially offering more consistent models for cancer studies and therapeutic development. Therefore, this technological advance fills a critical gap between the power of CRISPR technology and high-fidelity mouse models for studying human tumor evolution and preclinical drug testing.


Asunto(s)
Edición Génica , Neoplasias , Animales , Ratones , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Neoplasias/genética , Neoplasias/terapia , Reparación del ADN por Recombinación , Modelos Animales de Enfermedad
2.
Cancer Prev Res (Phila) ; 16(2): 65-73, 2023 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-36343340

RESUMEN

Antiestrogen medication is the only chemoprevention currently available for women at a high risk of developing breast cancer; however, antiestrogen therapy requires years to achieve efficacy and has adverse side effects. Therefore, it is important to develop an efficacious chemoprevention strategy that requires only a short course of treatment. PIK3CA is commonly activated in breast atypical hyperplasia, the known precancerous precursor of breast cancer. Targeting PI3K signaling in these precancerous lesions may offer a new strategy for chemoprevention. Here, we first established a mouse model that mimics the progression from precancerous lesions to breast cancer. Next, we demonstrated that a short-course prophylactic treatment with the clinically approved PI3K inhibitor alpelisib slowed early lesion expansion and prevented cancer formation in this model. Furthermore, we showed that alpelisib suppressed ex vivo expansion of patient-derived atypical hyperplasia. Together, these data indicate that the progression of precancerous breast lesions heavily depends on the PI3K signaling, and that prophylactic targeting of PI3K activity can prevent breast cancer. PREVENTION RELEVANCE: PI3K protein is abnormally high in breast precancerous lesions. This preclinical study demonstrates that the FDA-approved anti-PI3K inhibitor alpelisib can prevent breast cancer and thus warrant future clinical trials in high-risk women.


Asunto(s)
Lesiones Precancerosas , Tiazoles , Animales , Ratones , Femenino , Hiperplasia/tratamiento farmacológico , Tiazoles/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Lesiones Precancerosas/tratamiento farmacológico , Moduladores de los Receptores de Estrógeno , Fosfatidilinositol 3-Quinasa Clase I
3.
Oncogene ; 41(48): 5214-5222, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36261627

RESUMEN

Signal transducer and activator of transcription 5 (STAT5) promotes cell survival and instigates breast tumor formation, and in the normal breast it also drives alveolar differentiation and lactogenesis. However, whether STAT5 drives a differentiated phenotype in breast tumorigenesis and therefore impacts cancer spread and metastasis is unclear. We found in two genetically engineered mouse models of breast cancer that constitutively activated Stat5a (Stat5aca) caused precancerous mammary epithelial cells to become lactogenic and evolve into tumors with diminished potential to metastasize. We also showed that STAT5aca reduced the migratory and invasive ability of human breast cancer cell lines in vitro. Furthermore, we demonstrated that STAT5aca overexpression in human breast cancer cells lowered their metastatic burden in xenografted mice. Moreover, RPPA, Western blotting, and studies of ChIPseq data identified several EMT drivers regulated by STAT5. In addition, bioinformatic studies detected a correlation between STAT5 activity and better prognosis of breast cancer patients. Together, we conclude that STAT5 activation during mammary tumorigenesis specifies a tumor phenotype of lactogenic differentiation, suppresses EMT, and diminishes potential for subsequent metastasis.


Asunto(s)
Neoplasias de la Mama , Factor de Transcripción STAT5 , Animales , Femenino , Humanos , Ratones , Mama/patología , Neoplasias de la Mama/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/patología , Factor de Transcripción STAT5/metabolismo
4.
Clin Cancer Res ; 23(14): 3896-3905, 2017 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-28167508

RESUMEN

Purpose: A role of Dicer, which converts precursor miRNAs to mature miRNAs, in the tumor-promoting effect of hypoxia is currently emerging in some tumor entities. Its role in hepatocellular carcinoma (HCC) is unknown.Experimental Design: HepG2 and Huh-7 cells were stably transfected with an inducible Dicer expression vector and were exposed to hypoxia/normoxia. HepG2-Dicer xenografts were established in nude mice; hypoxic areas and Dicer were detected in HCC xenografts and HCCs from mice with endogenous hepatocarcinogenesis; and epithelial-mesenchymal transition (EMT) markers were analyzed by immunohistochemistry or by immunoblotting. The correlation between Dicer and carbonic anhydrase 9 (CA9), a marker of hypoxia, was investigated in resected human HCCs.Results: Hypoxia increased EMT markers in vitro and in vivo and led to a downregulation of Dicer in HCC cells. The levels of Dicer were downregulated in hypoxic tumor regions in mice with endogenous hepatocarcinogenesis and in HepG2 xenografts. In human HCCs, the levels of Dicer correlated inversely with those of CA9, indicating that the negative regulation of Dicer by hypoxia also applies to HCC patients. Forced expression of Dicer prevented the hypoxia-induced increase in hypoxia-inducible factor 1α (HIF1α), HIF2α, hypoxia-inducible genes (CA9, glucose transporter 1), EMT markers, and cell migration.Conclusions: We here identify downmodulation of Dicer as novel essential process in hypoxia-induced EMT in HCC and demonstrate that induced expression of Dicer counteracted hypoxia-induced EMT. Thus, targeting hypoxia-induced downmodulation of Dicer is a promising novel strategy to reduce HCC progression. Clin Cancer Res; 23(14); 3896-905. ©2017 AACR.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma Hepatocelular/genética , ARN Helicasas DEAD-box/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Hepáticas/genética , Ribonucleasa III/genética , Animales , Carcinogénesis/genética , Carcinoma Hepatocelular/patología , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Ratones , Hipoxia Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Transl Res ; 169: 40-6, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26608461

RESUMEN

MicroRNAs (miRNAs) circulating extracellularly in the blood are currently intensively studied as novel disease markers. However, the preanalytical factors influencing the levels of the extracellular miRNAs are still incompletely explored. In particular, it is unknown, whether the incubation of blood samples as occurring in clinical routine can lead to a release of miRNAs from blood cells and thus alter the extracellular miRNA levels before the preparation of serum or plasma from the blood cells. Using a set of marker miRNAs and quantitative RT-PCR, we found that the levels of extracellular miRNA-1, miRNA-16, and miRNA-21 were increased in EDTA and serum collection tubes incubated for 1-3 hours at room temperature and declined thereafter; the levels of the liver-specific miRNA-122 declined monophasically. These events occurred in the absence of significant hemolysis. When the blood was supplemented with Ribonuclease A inhibitor, the levels of miRNA-1, miRNA-16, and miRNA-21 increased substantially during the initial 3 hours of incubation and those of miRNA-122 remained unchanged, indicating that the release of blood cell-derived miRNAs occurred during the initial 3 hours of incubation of the blood tubes, but not at later time points. Separation of 5-hour preincubated blood into vesicle and nonvesicle fractions revealed a selective increase in the portion of vesicle-associated miRNAs. Together, these data indicate that the release of vesicle-associated miRNAs from blood cells can occur in blood samples within the time elapsing in normal clinical practice until their processing without significant hemolysis. This becomes particularly visible on the inhibition of miRNA degradation by Ribonuclease A inhibitor.


Asunto(s)
Células Sanguíneas/metabolismo , MicroARNs/sangre , Humanos
6.
Cancer Res ; 75(15): 3147-54, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-26239478

RESUMEN

iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA-enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA-enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors.


Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Doxorrubicina/administración & dosificación , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Oligopéptidos/administración & dosificación , Compuestos de Fenilurea/administración & dosificación , Administración Intravenosa , Secuencias de Aminoácidos , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Doxorrubicina/farmacocinética , Sistemas de Liberación de Medicamentos , Azul de Evans/administración & dosificación , Gadolinio DTPA , Células Hep G2/efectos de los fármacos , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones Desnudos , Ratones Transgénicos , Niacinamida/administración & dosificación , Oligopéptidos/química , Sorafenib , Distribución Tisular
7.
J Infect Dev Ctries ; 4(4): 226-34, 2010 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-20440060

RESUMEN

BACKGROUND: This work demonstrates successful propagation of HCV in HepG2 and human blood cells as well as viral shedding into their culture media. The influence of Schistosoma mansoni crude soluble egg antigens (SEA) on the rate of viral propagation in both mammalian cells was also monitored. METHODOLOGY: HepG2 cells were inoculated with HCV viremic human sera and some wells were exposed to HCV infection in presence of SEA. Cells were harvested for RT-PCR and Western blotting analysis. HepG2 media was collected for HCV ELISA. Blood samples from HCV-infected humans were cultured in the presence and absence of SEA. Media were collected at different time points post culturing and subjected to HCV ELISA. RESULTS: The ELISA concentration of HCV antigens were generally higher in media of infected HepG2 cells compared to media of control cells at all time intervals post infection. Western blots showed reactivity to immunogenic peptides of different molecular weights in lysate of infected HepG2 cells that were not evidenced in uninfected cells. In presence of SEA, RT-PCR results revealed earlier detection of viral RNA in infected HepG2 cells compared to in absence of such bilharzial antigen. Also, ELISA results revealed higher levels of detected HCV antigens in media of both infected HepG2 and blood cells cocultured with S. mansoni SEA compared to that of cultured infected cells in absence of the parasite antigens. CONCLUSION: HepG2 cells as well as whole blood cultures maintain HCV replication. Furthermore, SEA has the potential to enhance HCV propagation.


Asunto(s)
Antígenos Helmínticos/aislamiento & purificación , Hepacivirus/efectos de los fármacos , Hepacivirus/crecimiento & desarrollo , Péptidos y Proteínas de Señalización Intercelular/aislamiento & purificación , Schistosoma mansoni/química , Replicación Viral/efectos de los fármacos , Animales , Antígenos Virales/análisis , Antígenos Virales/inmunología , Western Blotting , Línea Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Arch Virol ; 154(10): 1649-57, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19763775

RESUMEN

The role of the NS3 protease in HCV replication was demonstrated by the ability of a protease inhibitor cocktail (10 microg/ml) to abolish the induced cytopathic effect in RAW macrophages upon infection with Egyptian sera. The HCV protease gene was amplified from Egyptian sera by nested PCR and cloned downstream of the CMV promotor in a mammalian expression plasmid, which was then used to transform bacteria. Colonies carrying the gene in the correct orientation were subjected to large-scale plasmid purification followed by sequencing. Phylogenetic comparison of the sequence obtained with published sequences from different genotypes confirmed that our sequence belongs to genotype 4a. Of the other genotypes, the most closely related ones were from genotype 1. Multiple alignments of protease peptides showed that the catalytic triads and binding residues for substrate, Zn2+ and the NS4 cofactor are conserved among different isolates, including ours, and confirmed the closer homology between NS3 of genotypes 4 and 1. The HCV-protease-encoding construct was successfully transcribed in both mammalian cells and mice. Mouse antibodies produced against the protease-encoding-construct detected the 18-kDa enzyme in lysates of cells transfected with the construct by Western blotting, and in the media of infected cells by ELISA.


Asunto(s)
Hepacivirus/enzimología , Proteínas no Estructurales Virales/fisiología , Animales , Línea Celular , Clonación Molecular , Egipto , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación Viral de la Expresión Génica/fisiología , Genotipo , Hepacivirus/genética , Hepatitis C/virología , Humanos , Macrófagos/virología , Ratones , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Proteínas no Estructurales Virales/genética , Replicación Viral/genética , Replicación Viral/fisiología
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