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1.
Nat Commun ; 12(1): 1453, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674603

RESUMEN

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.


Asunto(s)
Adenocarcinoma/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Inmunoterapia/métodos , Neoplasias Pancreáticas/metabolismo , Tetraspaninas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/terapia , Animales , Antígenos de Neoplasias/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/terapia , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Citocinas/metabolismo , Proteínas Ligadas a GPI/metabolismo , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Factores Inmunológicos , Activación de Linfocitos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Linfocitos T/inmunología , Tetraspaninas/genética
2.
Nat Commun ; 12(1): 1956, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782403

RESUMEN

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c+). NPM1c+ maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c+ controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c+-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c+-driven leukemogenesis by rectifying the signature of NPM1c+ leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1c/+) mice. HoxBlincTg and Npm1c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c+ signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c+ leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c+ AML.


Asunto(s)
Carcinogénesis/genética , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , ARN Largo no Codificante/genética , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Perfilación de la Expresión Génica , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Transgénicos , Familia de Multigenes , Mutación , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Mielopoyesis/genética , Proteínas Nucleares/deficiencia , Regiones Promotoras Genéticas , ARN Largo no Codificante/agonistas , ARN Largo no Codificante/metabolismo , Transducción de Señal , Transcripción Genética
3.
Methods Mol Biol ; 2279: 165-173, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683693

RESUMEN

Patient-derived xenografts (PDXs) are created by implanting human tumor tissue or cells into immunodeficent mice, and enable the study of tumor biology, biomarkers and response to therapy in vivo. This chapter describes a method for lung adenocarcinoma (LAC) PDX generation using subcutaneous implantation of tumor tissue and cell suspensions and incorporating the humanization of PDX models by reconstitution with human immune cells.


Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Trasplante de Neoplasias , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Femenino , Xenoinjertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID
4.
Nat Commun ; 12(1): 1623, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712589

RESUMEN

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Metástasis de la Neoplasia , Fosfatidilinositoles/metabolismo , Transducción de Señal , Factores de Ribosilacion-ADP/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular Tumoral , Factores de Intercambio de Guanina Nucleótido/genética , Xenoinjertos , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo
5.
Nat Commun ; 12(1): 1628, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712615

RESUMEN

Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents.


Asunto(s)
Resistencia a Antineoplásicos/genética , Tolerancia a Medicamentos/genética , Tolerancia a Medicamentos/fisiología , Neoplasias/genética , Neoplasias/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , Combinación de Medicamentos , Descubrimiento de Drogas , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/efectos de los fármacos , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células U937
6.
Nat Commun ; 12(1): 1156, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33608544

RESUMEN

Cancer immunoediting is a dynamic process of crosstalk between tumor cells and the immune system. Herein, we explore the fast zebrafish xenograft model to investigate the innate immune contribution to this process. Using multiple breast and colorectal cancer cell lines and zAvatars, we find that some are cleared (regressors) while others engraft (progressors) in zebrafish xenografts. We focus on two human colorectal cancer cells derived from the same patient that show contrasting engraftment/clearance profiles. Using polyclonal xenografts to mimic intra-tumor heterogeneity, we demonstrate that SW620_progressors can block clearance of SW480_regressors. SW480_regressors recruit macrophages and neutrophils more efficiently than SW620_progressors; SW620_progressors however, modulate macrophages towards a pro-tumoral phenotype. Genetic and chemical suppression of myeloid cells indicates that macrophages and neutrophils play a crucial role in clearance. Single-cell-transcriptome analysis shows a fast subclonal selection, with clearance of regressor subclones associated with IFN/Notch signaling and escaper-expanded subclones with enrichment of IL10 pathway. Overall, our work opens the possibility of using zebrafish xenografts as living biomarkers of the tumor microenvironment.


Asunto(s)
Neoplasias del Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Evasión Inmune , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Xenoinjertos , Proteínas de Homeodominio/genética , Humanos , Inmunidad Innata , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra
7.
J Vis Exp ; (167)2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33586709

RESUMEN

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the "Murine Ommaya." As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.


Asunto(s)
Enfermedades del Sistema Nervioso Central/terapia , Sistemas de Liberación de Medicamentos , Xenoinjertos/fisiología , Modelos Biológicos , Animales , Neoplasias de la Mama/patología , Femenino , Inyecciones Intraventriculares , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/tratamiento farmacológico , Neoplasias Meníngeas/patología , Ratones , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/patología , Pronóstico
8.
Nat Commun ; 12(1): 1302, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33637726

RESUMEN

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.


Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma , Proteínas/genética , Animales , Apoptosis , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Técnicas de Inactivación de Genes , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Unión al ARN/genética , Transcriptoma
9.
Nature ; 591(7850): 451-457, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33561864

RESUMEN

All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.


Asunto(s)
/tratamiento farmacológico , Citidina/análogos & derivados , Hidroxilaminas/administración & dosificación , Hidroxilaminas/uso terapéutico , Administración Oral , Células Epiteliales Alveolares/inmunología , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , Animales , Quimioprevención , Quirópteros/virología , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Citidina/administración & dosificación , Citidina/uso terapéutico , Citocinas/inmunología , Células Epiteliales/virología , Femenino , Xenoinjertos , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Trasplante de Pulmón , Masculino , Ratones , Profilaxis Posexposición , Profilaxis Pre-Exposición , /patogenicidad , Replicación Viral
11.
Nat Commun ; 12(1): 192, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33420019

RESUMEN

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.


Asunto(s)
Carcinogénesis/metabolismo , Diferenciación Celular , Proteína MioD/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Rabdomiosarcoma/genética , Rabdomiosarcoma/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Animales , Carcinogénesis/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Factores de Transcripción MEF2/metabolismo , Masculino , Ratones , Ratones SCID , Desarrollo de Músculos/genética , Proteína MioD/genética , Miogenina/metabolismo , Proteínas de Fusión Oncogénica/genética , Oncogenes , Rabdomiosarcoma/patología , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Embrionario/genética , Factores de Transcripción de la Familia Snail/genética , Transcriptoma
12.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-33430352

RESUMEN

The therapeutic benefit of immune checkpoint inhibitor monotherapy is limited to a subset of patients in urothelial carcinoma (UC). Previous studies showed the immunogenicity of cisplatin and irradiation. Here, we investigated whether chemoradiotherapy (CRT), a combination of cisplatin and irradiation, could improve the efficacy of postirradiation anti-programmed cell death 1 (PD-1) treatment in UC. In our advanced UC patient cohort, patients with CRT showed a significantly better objective response rate (75%/22%) and overall survival (88%/30% at 12 months) following later pembrolizumab therapy compared to those without. Then, we created syngeneic UC mouse models by inoculating MB49 cells s.c. in C57BL/6J mice to examine the potential of CRT to enhance antitumor immunity in conjunction with postirradiation anti-PD-1 treatment. Nonirradiated tumors of the mice treated with CRT/postirradiation anti-PD-1 treatment had a significantly slower growth rate and a significantly higher expression of cytotoxic T cells compared to those of the mice treated with anti-PD-1 treatment alone. The mice treated with CRT/postirradiation anti-PD-1 treatment showed the best survival. Mechanistically, CRT provoked strong direct cytotoxicity and increased expressions of immunogenic cell death markers in MB49 cells. Therefore, the combination of cisplatin and irradiation induces immunogenic cell death and potentiates postirradiation anti-PD-1 treatment efficacy in UC.


Asunto(s)
Carcinoma/tratamiento farmacológico , Carcinoma/radioterapia , Cisplatino/farmacología , Muerte Celular Inmunogénica/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos/farmacología , Carcinoma/genética , Carcinoma/patología , Quimioradioterapia , Terapia Combinada , Xenoinjertos , Humanos , Ratones , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Urotelio/efectos de los fármacos , Urotelio/patología , Urotelio/efectos de la radiación
13.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-33430361

RESUMEN

Anaplastic thyroid cancer (ATC) is an undifferentiated and advanced form of thyroid cancer, accompanied with a high ratio of epigenetic adjustment, which occurs more than genetic mutations. In this study, we aimed to evaluate the synergistic anticancer effect (in vitro and in vivo) of the new combination of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) and sorafenib with radiation therapy in pre-clinical models of ATC. The ATC cell lines, YUMC-A1 and YUMC-A2, were isolated from the current patients who were treated with HNHA and sorafenib, either as monotherapy or combination therapy. Synergistic anticancer effect of the combination therapy on the intracellular signaling pathways and cell cycle was assessed via flow cytometry and immunoblot analysis. To examine tumor shrinkage activity in vivo, an ATC cell line-derived mouse xenograft model was used. Results showed that the combination therapy of HNHA and sorafenib with radiation promoted tumor suppression via caspase cleavage and cell cycle arrest in patient-derived ATC. In addition, the combination therapy of HNHA and sorafenib with radiation was more effective against ATC than therapy with HNHA or sorafenib with radiation. Thus, the combination of HNHA and sorafenib with radiation may be used as a novel curative approach for the treatment of ATC.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Naftalenos/farmacología , Sorafenib/farmacología , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/radioterapia , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Terapia Combinada , Sinergismo Farmacológico , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Radioterapia , Carcinoma Anaplásico de Tiroides/patología
14.
J Cancer Res Clin Oncol ; 147(3): 703-712, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33386469

RESUMEN

OBJECTIVE: The malignant transformation of normal bladder cells (SV-HUC-1) was induced by arsenite to explore the possible mechanism of circRNA-100284 influencing bladder cancer cell proliferation. METHODS: Normal bladder SV-HUC-1 cells were cultured with 2 µM arsenite to induce malignant transformation. After 0, 3, 6, 12, and 24 h of culture, the expression level of circRNA-100284 in cells was detected by quantitative real-time PCR. Western blotting assays were used to detect the expression levels of EZH2 and cyclin-D1 proteins in cells treated with different media. Cell cycle was analyzed by flow cytometry. In addition, through cell transfection and CCK-8 experiments, the effect and mechanism of circRNA-100284 targeting microRNA-217 on proliferation was determined. The interaction between HSP70 methylation and Aurora-B was determined by Western blotting and immunoprecipitation experiments. RESULTS: With prolonged contact time with arsenite, the expression level of circRNA-100284 in cells increased continuously (P < 0.05). Western blotting assays showed that the expression levels of EZH2 and cyclin-D1 proteins in arsenite-transformed cells increased. Flow cytometry and CCK-8 showed that circRNA-100284 accelerated cell cycle transition and cell proliferation through miR-217. Finally, after culturing human bladder cancer T24 cells, combined with immunoprecipitation and in vitro kinase experiments, it was found that K561- dimethyl HSP70 activated Aurora-B, thus promoting the proliferation of bladder cancer cells. CONCLUSION: CircRNA-100284 activates aurora kinase B by inducing methylation of HSP70 via microRNA-217 to promote the proliferation of bladder cancer cells.


Asunto(s)
Aurora Quinasa B/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , MicroARNs/metabolismo , ARN Circular/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Animales , Arsenitos/farmacología , Aurora Quinasa B/genética , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Transformación Celular Neoplásica/inducido químicamente , Ciclina D2/genética , Ciclina D2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Activación Enzimática , Femenino , Proteínas HSP70 de Choque Térmico/genética , Xenoinjertos , Humanos , Metilación , Ratones , MicroARNs/genética , ARN Circular/genética , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología
15.
Methods Mol Biol ; 2206: 151-178, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32754817

RESUMEN

This protocol focuses on the quantitative description of the angioarchitecture of experimental tumor xenografts. This semiautomatic analysis is carried out on functional vessels and microvessels acquired by confocal imaging and processed into progressively reconstructed angioarchitectures following a caliber-classification step. The protocol can be applied also to the quantification of pathological angioarchitectures other than tumor grafts as well as to the microvasculature of physiological tissue samples.


Asunto(s)
Microscopía Confocal/métodos , Microvasos/patología , Neoplasias/patología , Neovascularización Patológica/patología , Animales , Xenoinjertos/patología , Humanos , Ratones
16.
Methods Mol Biol ; 2206: 179-192, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32754818

RESUMEN

Xenograft models allow for an in vivo approach to monitor cellular functions within the context of a host microenvironment. Here we describe a protocol to generate a xenograft model of venous malformation (VM) based on the use of human umbilical vein endothelial cells (HUVEC) expressing a constitutive active form of the endothelial tyrosine kinase receptor TEK (TIE2 p.L914F) or patient-derived EC containing TIE2 and/or PIK3CA gene mutations. Hyperactive somatic TIE2 and PIK3CA mutations are a common hallmark of VM in patient lesions. The EC are injected subcutaneously on the back of athymic nude mice to generate ectatic vascular channels and recapitulate histopathological features of VM patient tissue histology. Lesion plugs with TIE2/PIK3CA-mutant EC are visibly vascularized within 7-9 days of subcutaneous injection, making this a great tool to study venous malformation.


Asunto(s)
Xenoinjertos/patología , Células Endoteliales de la Vena Umbilical Humana/patología , Malformaciones Vasculares/patología , Venas/patología , Animales , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Modelos Animales de Enfermedad , Xenoinjertos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Ratones Desnudos , Receptor TIE-2/metabolismo , Malformaciones Vasculares/metabolismo , Venas/metabolismo
17.
Nature ; 590(7846): 498-503, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33361816

RESUMEN

Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family, including NSD1, NSD2 and NSD3, have crucial roles in chromatin regulation and are implicated in oncogenesis1,2. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling dimethylation of histone H3 at Lys36 (H3K36)3-7. However, the molecular basis that underlies this mechanism is largely unknown. Here we solve the cryo-electron microscopy structures of NSD2 and NSD3 bound to mononucleosomes. We find that binding of NSD2 and NSD3 to mononucleosomes causes DNA near the linker region to unwrap, which facilitates insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between NSD2 or NSD3 and the nucleosome precisely defines the position of the enzyme on the nucleosome, explaining the specificity of methylation to H3K36. Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3. NSDs that contain these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes the proliferation of cancer cells and the growth of xenograft tumours. Together, our research provides molecular insights into the nucleosome-based recognition and histone-modification mechanisms of NSD2 and NSD3, which could lead to strategies for therapeutic targeting of proteins of the NSD family.


Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Proteínas Represoras/metabolismo , Sitios de Unión , Biocatálisis , Línea Celular Tumoral , Proliferación Celular , Microscopía por Crioelectrón , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/ultraestructura , Histonas/ultraestructura , Humanos , Metilación , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestructura , Nucleosomas/ultraestructura , Fenotipo , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/ultraestructura
18.
Life Sci ; 267: 118933, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33359744

RESUMEN

AIMS: Non-small cell lung cancer (NSCLC) is considered a highly fatal tumor. Importantly, angiogenesis is critical for tumor progression. Long non-coding RNAs (lncRNAs), which are untranslatable, control cell functions through different pathways. lncRNA EPIC1 has been reported to promote cell viability, cell cycle progression, and invasion. However, the relationship between EPIC1 and tumor angiogenesis remains an enigma. We explored the role of EPIC1 in tumor angiogenesis in NSCLC. MATERIALS AND METHODS: First, EPIC1 expression was analyzed using the GEPIA database and was further verified using qPCR in tumor tissues from patients with NSCLC and NSCLC cell lines. Next, EPIC1 function was detected using loss-of-function and gain-of-function assays. Moreover, EdU staining, flow cytometry, and channel formation assays were performed to assess HUVEC proliferation and channel the formation in the NSCLC-HUVEC transwell co-culture system. KEY FINDINGS: EPIC1 expression was significantly upregulated in NSCLC tissues and cell lines. Furthermore, the overexpression of EPIC1 in NSCLC cells stimulated HUVEC channel formation and proliferation by activating Ang2/Tie2 signaling, and the opposite results were obtained when EPIC1 was silenced in NSCLC cells. The density of new blood vessels was simultaneously increased by EPIC1 overexpression in vivo, using CAM angiogenesis model and a nude mouse tumor model. Finally, all these experimental findings could be established in the samples from patients with NSCLC. We postulate that EPIC1 promotes tumor angiogenesis by activating the Ang2/Tie2 axis in NSCLC. SIGNIFICANCE: Elucidating the molecular and cellular mechanisms of EPIC1 in tumor angiogenesis provides a novel perspective on NSCLC clinical therapy.


Asunto(s)
Angiopoyetina 2/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , ARN Largo no Codificante/genética , Receptor TIE-2/metabolismo , Angiopoyetina 2/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Embrión de Pollo , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Xenoinjertos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Patológica/genética , Neovascularización Patológica/patología , ARN Largo no Codificante/metabolismo , Receptor TIE-2/genética , Transducción de Señal
19.
Am J Pathol ; 191(2): 385-395, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33321090

RESUMEN

Insulin-induced gene 2 (INSIG2) functions as a blocker of cholesterol biosynthesis and has been shown to be involved in colon and pancreatic cancer pathogenesis. Cholesterol is a risk factor for breast cancer pathophysiology; however, the underlying mechanisms are not well-defined. Hence, our goal was to determine the role of INISG2 in breast cancer. INSIG2 mRNA and protein expression was correlated to metastatic potential of breast cancer cell lines. Knockdown of INSIG2 inhibited epithelial-to-mesenchymal transition. Conversely, overexpression of INSIG2 induced epithelial-to-mesenchymal transition. Knockdown of INSIG2 did not affect cell proliferation but resulted in altered metabolism in vitro and attenuated experimental metastasis in vivo. Analysis of breast cancer tissue microarrays revealed significantly higher INSIG2 protein expression in breast cancer tissues. INSIG2 protein expression was correlated to hormone receptor status, with significantly higher expression in patients with triple-negative and human epidermal growth factor receptor 2 molecular subtypes of invasive breast cancer. Analysis of The Cancer Genome Atlas, however, revealed significantly lower INSIG2 mRNA expression in triple-negative breast cancer patients. Higher INSIG2 mRNA expression was correlated to poor survival probability. Asian patients with high INSIG2 mRNA expression had significantly lower survival probability compared with Asian patients with low/medium INSIG2 mRNA expression. These results reveal a yet undefined role of INSIG2 in breast cancer, potentially more relevant for breast cancer patients in Asia.


Asunto(s)
Neoplasias de la Mama/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Metástasis de la Neoplasia/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/patología
20.
Microvasc Res ; 133: 104072, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32949573

RESUMEN

BACKGROUND: The process of angiogenesis is a key element for tumor growth and proliferation and therefore one of the determining factors for aggressiveness and malignancy. A better understanding of the underlying processes of tumor induced angiogenesis is crucial for superior cancer treatment. Furthermore, the PeriCam perfusion speckle imager (PSI) system high resolution (HR) model by PERIMED presents a noninvasive method for semi-quantitative measurement of blood perfusion, based on laser speckle contrast analysis (LASCA). Aim of the present study was to utilize the chick chorioallantoic membrane (CAM) model as an in-ovo-tumor-model which enables rapid neovascularization of tumors while allowing real-time observation of the microcirculation via LASCA. METHODS: Fertilized chicken eggs were grafted with embryonal/alveolar rhabdomyosarcoma cells or primary sarcoma tumors. The blood perfusion was measured before and after tumor growth using LASCA. The procedure is accelerated and simplified through the integrated PIMSoft software which provides real-time graphs and color-coded images during the measurement. RESULTS: Sarcoma cells and primary sarcoma tumors exhibited satisfactory growth processes on the CAM. LASCA visualized microcirculation accurately and enabled an extensive investigation of the angiogenic potential of sarcoma cells on the CAM. We were able to show that sarcoma cells and primary sarcoma tumors induced larger quantities of neovasculature on the CAM than the controls. CONCLUSIONS: The utilization of LASCA for the investigation of tumor angiogenesis within the CAM model appears to be a highly beneficial, cost-efficient and easily practicable procedure. The proposed model can be used as a drug-screening model for individualized cancer therapy, especially with regards to anti-angiogenic agents.


Asunto(s)
Membrana Corioalantoides/irrigación sanguínea , Flujometría por Láser-Doppler , Neovascularización Patológica , Imagen de Perfusión , Rabdomiosarcoma Alveolar/irrigación sanguínea , Rabdomiosarcoma Embrionario/irrigación sanguínea , Sarcoma/irrigación sanguínea , Animales , Velocidad del Flujo Sanguíneo , Línea Celular Tumoral , Embrión de Pollo , Xenoinjertos , Humanos , Flujo Sanguíneo Regional , Factores de Tiempo , Carga Tumoral , Células Tumorales Cultivadas
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