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1.
Br J Cancer ; 122(4): 517-527, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31844184

RESUMEN

BACKGROUND: Docetaxel chemotherapy in prostate cancer has a modest impact on survival. To date, efforts to develop combination therapies have not translated into new treatments. We sought to develop a novel therapeutic strategy to tackle chemoresistant prostate cancer by enhancing the efficacy of docetaxel. METHODS: We performed a drug-repurposing screen by using murine-derived prostate cancer cell lines driven by clinically relevant genotypes. Cells were treated with docetaxel alone, or in combination with drugs (n = 857) from repurposing libraries, with cytotoxicity quantified using High Content Imaging Analysis. RESULTS: Mebendazole (an anthelmintic drug that inhibits microtubule assembly) was selected as the lead drug and shown to potently synergise docetaxel-mediated cell killing in vitro and in vivo. Dual targeting of the microtubule structure was associated with increased G2/M mitotic block and enhanced cell death. Strikingly, following combined docetaxel and mebendazole treatment, no cells divided correctly, forming multipolar spindles that resulted in aneuploid daughter cells. Liposomes entrapping docetaxel and mebendazole suppressed in vivo prostate tumour growth and extended progression-free survival. CONCLUSIONS: Docetaxel and mebendazole target distinct aspects of the microtubule dynamics, leading to increased apoptosis and reduced tumour growth. Our data support a new concept of combined mebendazole/docetaxel treatment that warrants further clinical evaluation.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Docetaxel/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Mebendazol/farmacología , Neoplasias de la Próstata , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Reposicionamiento de Medicamentos/métodos , Sinergismo Farmacológico , Humanos , Masculino , Ratones , Células PC-3 , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Development ; 144(1): 74-82, 2017 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-27888192

RESUMEN

Macrophages are important regulators of branching morphogenesis during development and postnatally in the mammary gland. Regulation of macrophage dynamics during these processes can therefore have a profound impact on development. We demonstrate here that the developing mammary gland expresses high levels of inflammatory CC-chemokines, which are essential in vivo regulators of macrophage migration. We further demonstrate that the atypical chemokine receptor ACKR2, which scavenges inflammatory CC-chemokines, is differentially expressed during mammary gland development. We have previously shown that ACKR2 regulates macrophage dynamics during lymphatic vessel development. Here, we extend these observations to reveal a novel role for ACKR2 in regulating the postnatal development of the mammary gland. Specifically, we show that Ackr2-/- mice display precocious mammary gland development. This is associated with increased macrophage recruitment to the developing gland and increased density of the ductal epithelial network. These data demonstrate that ACKR2 is an important regulator of branching morphogenesis in diverse biological contexts and provide the first evidence of a role for chemokines and their receptors in postnatal development processes.


Asunto(s)
Glándulas Mamarias Animales/embriología , Morfogénesis/genética , Receptores CCR/fisiología , Receptores de Quimiocina/fisiología , Animales , Movimiento Celular/genética , Embrión de Mamíferos , Femenino , Linfangiogénesis/genética , Vasos Linfáticos/embriología , Vasos Linfáticos/fisiología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células del Estroma/metabolismo
3.
Cytotherapy ; 22(12): 762-771, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32828673

RESUMEN

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs). METHODS: MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs. RESULTS: MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions. CONCLUSIONS: LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.


Asunto(s)
Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula/métodos , Inmunidad , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Neovascularización Fisiológica , Páncreas/citología , Biomarcadores/metabolismo , Diferenciación Celular , Proliferación Celular , Forma de la Célula , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Humanos , Inmunomodulación , Interferón gamma/metabolismo , Medicina Regenerativa , Linfocitos T/citología
4.
J Biol Chem ; 289(18): 12330-42, 2014 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-24644289

RESUMEN

The atypical chemokine receptor, ACKR2 is a pivotal regulator of chemokine-driven inflammatory responses and works by binding, internalizing, and degrading inflammatory CC-chemokines. ACKR2 displays promiscuity of ligand binding and is capable of interacting with up to 14 different inflammatory CC-chemokines. Despite its prominent biological role, little is known about the structure/function relationship within ACKR2, which regulates ligand binding. Here we demonstrate that a conserved tyrosine motif at the N terminus of ACKR2 is essential for ligand binding, internalization, and scavenging. In addition we demonstrate that sulfation of this motif contributes to ligand internalization. Furthermore, a peptide derived from this region is capable of binding inflammatory chemokines and inhibits their interaction with their cognate signaling receptors. Importantly, the peptide is only active in the sulfated form, further confirming the importance of the sulfated tyrosines for function. Finally, we demonstrate that the bacterial protease, staphopain A, can cleave the N terminus of ACKR2 and suppress its ligand internalization activity. Overall, these results shed new light on the nature of the structural motifs in ACKR2 that are responsible for ligand binding. The study also highlights ACKR2-derived N-terminal peptides as being of potential therapeutic significance.


Asunto(s)
Secuencias de Aminoácidos , Quimiocinas/metabolismo , Receptores de Quimiocina/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Western Blotting , Células CHO , Línea Celular Tumoral , Células Cultivadas , Cricetinae , Cricetulus , Cisteína Endopeptidasas/metabolismo , Endocitosis/genética , Células HEK293 , Humanos , Ligandos , Datos de Secuencia Molecular , Mutación , Péptidos/metabolismo , Unión Proteica , Interferencia de ARN , Receptores de Quimiocina/química , Receptores de Quimiocina/genética , Homología de Secuencia de Aminoácido , Sulfatos/metabolismo , Tirosina/genética
5.
Blood ; 121(18): 3768-77, 2013 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-23479571

RESUMEN

The mechanisms by which CC chemokine receptor (CCR)7 ligands are selectively presented on lymphatic endothelium in the presence of inflammatory chemokines are poorly understood. The chemokine-scavenging receptor D6 is expressed on lymphatic endothelial cells (LEC) and contributes to selective presentation of CCR7 ligands by suppressing inflammatory chemokine binding to LEC surfaces. As well as preventing inappropriate inflammatory cell attachment to LECs, D6 is specifically involved in regulating the ability of LEC to discriminate between mature and immature dendritic cells (DCs). D6 overexpression reduces immature DC (iDC) adhesion to LECs, whereas D6 knockdown increases adhesion of iDCs that displace mature DCs. LEC D6 expression is regulated by growth factors, cytokines, and tumor microenvironments. In particular, interleukin-6 and interferon-γ are potent inducers, indicating a preferential role for D6 in inflamed contexts. Expression of the viral interleukin-6 homolog from Kaposi sarcoma-associated herpesvirus is also sufficient to induce significant D6 upregulation both in vitro and in vivo, and Kaposi sarcoma and primary effusion lymphoma cells demonstrate high levels of D6 expression. We therefore propose that D6, which is upregulated in both inflammatory and tumor contexts, is an essential regulator of inflammatory leukocyte interactions with LECs and is required for immature/mature DC discrimination by LECs.


Asunto(s)
Células Endoteliales/metabolismo , Receptores CCR10/genética , Receptores CCR10/fisiología , Animales , Células CHO , Comunicación Celular/genética , Comunicación Celular/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Cricetinae , Cricetulus , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/fisiología , Células Endoteliales/inmunología , Células HEK293 , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores CCR10/análisis , Receptores CCR10/metabolismo , Transfección , Receptor de Quimiocina D6
6.
Cancers (Basel) ; 11(5)2019 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-31032816

RESUMEN

Inducible genetically defined mouse models of cancer uniquely facilitate the investigation of early events in cancer progression, however, there are valid concerns about the ability of such models to faithfully recapitulate human disease. We developed an inducible mouse model of progressive lung adenocarcinoma (LuAd) that combines sporadic activation of oncogenic KRasG12D with modest overexpression of c-MYC (KM model). Histological examination revealed a highly reproducible spontaneous transition from low-grade adenocarcinoma to locally invasive adenocarcinoma within 6 weeks of oncogene activation. Laser-capture microdissection coupled with RNA-SEQ (ribonucleic acid sequencing) was employed to determine transcriptional changes associated with tumour progression. Upregulated genes were triaged for relevance to human LuAd using datasets from Oncomine and cBioportal. Selected genes were validated by RNAi screening in human lung cancer cell lines and examined for association with lung cancer patient overall survival using KMplot.com. Depletion of progression-associated genes resulted in pronounced viability and/or cell migration defects in human lung cancer cells. Progression-associated genes moreover exhibited strong associations with overall survival, specifically in human lung adenocarcinoma, but not in squamous cell carcinoma. The KM mouse model faithfully recapitulates key molecular events in human adenocarcinoma of the lung and is a useful tool for mechanistic interrogation of KRAS-driven LuAd progression.

7.
Cell Death Dis ; 9(11): 1069, 2018 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-30341281

RESUMEN

Based on a molecular classification of prostate cancer using gene expression pathway signatures, we derived a set of 48 genes in critical pathways that significantly predicts clinical outcome in all tested patient cohorts. We tested these genes in a functional genomics screen in a panel of three prostate cancer cell lines (LNCaP, PC3, DU145), using RNA interference. The screen revealed several genes whose knockdown caused strong growth inhibition in all cell lines. Additionally, we tested the gene set in the presence of docetaxel to see whether any gene exhibited additive or synergistic effects with the drug. We observed a strong synergistic effect between DLGAP5 knockdown and docetaxel in the androgen-sensitive line LNCaP, but not in the two other androgen-independent lines. We then tested whether this effect was connected to androgen pathways and found that knockdown of the androgen receptor by si-RNA attenuated the synergy significantly. Similarly, androgen desensitized LNCaP-AI cells had a higher IC50 to docetaxel and did not exhibit the synergistic interaction. Short-term exposure to enzalutamide did not significantly alter the behaviour of parental LNCaP cells. An immunofluorescence analysis in LNCaP cells suggests that under the double insult of DLGAP5 knockdown and docetaxel, cells predominantly arrest in metaphase. In contrast, the knockdown of the androgen receptor by siRNA appears to assist cells to progress through metaphase in to anaphase, even in the presence of docetaxel. Our data suggest that DLGAP5 has a unique function in stabilizing spindle formation and surviving microtubule assault from docetaxel, in an androgen-regulated cell cycle system.


Asunto(s)
Docetaxel/farmacología , Genómica/métodos , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Receptores Androgénicos/metabolismo , Benzamidas , Proteínas Cdc20/genética , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Metafase , Proteínas Asociadas a Microtúbulos/genética , Nitrilos , Células PC-3 , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Receptores Androgénicos/genética , Transfección
8.
J Natl Cancer Inst ; 110(5): 467-478, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29165716

RESUMEN

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


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Autofagia/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib/administración & dosificación , Imidazoles/administración & dosificación , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Terapia Molecular Dirigida/métodos , Piridazinas/administración & dosificación , Pirimidinas/administración & dosificación , Quinolinas/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto
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