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1.
Int J Cancer ; 145(7): 1991-2001, 2019 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30848481

RESUMEN

Sunitinib is one of the most widely used targeted therapeutics for renal cell carcinoma (RCC), but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in RCC, we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and after development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in silico prediction models, six predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1, and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function renders tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the six proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.


Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma de Células Renales/genética , Resistencia a Antineoplásicos , Neoplasias Renales/genética , Mutación , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Exones , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/metabolismo , Ratones , Trasplante de Neoplasias , Análisis de Secuencia de ADN , Sunitinib
2.
Eur J Haematol ; 97(2): 128-36, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26505263

RESUMEN

OBJECTIVE: The aim of this study was to assess differences in the gene expression profile of peripheral blood cells between patients with early recurrent thrombosis vs. patients without recurrent events after withdrawal of anticoagulant therapy for a first episode of unprovoked deep vein thrombosis (uDVT), to identify novel predictors of recurrence. METHODS: In the discovery population (N = 32), a microarray RNA assay followed by RT-PCR confirmation were performed. In the validation population (N = 44) a multiple RT-PCR-based strategy was applied to assess genes differentially expressed in the discovery population. RESULTS: The sex-adjusted Linear Model for Microarray Data analysis showed 102 genes differentially expressed (P < 0.01) in the discovery population. Nineteen of them underwent further confirmation in the validation population. The gene encoding for Acyl-CoA Synthetase Family Member 2 (ACSF2) was underexpressed in recurrent DVT patients in both, the discovery (P = 0.007) and validation populations (P = 0.004). In the receiver operator characteristic (ROC) analysis, the areas under the curve of ACSF2 expression were 0.77 and 0.80, respectively. CONCLUSIONS: For the first time an association between ACSF2 expression and the risk of recurrent DVT is suggested. Should this association be confirmed in larger prospective studies, ACSF2 could become useful for the selection of patients requiring extended anticoagulant therapy.


Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Trombosis de la Vena/genética , Trombosis de la Vena/patología , Adulto , Biomarcadores , Análisis por Conglomerados , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Recurrencia , Estudios Retrospectivos , Factores de Riesgo , Sensibilidad y Especificidad , Trombosis de la Vena/diagnóstico
3.
JHEP Rep ; 6(8): 101118, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39105183

RESUMEN

Background & Aims: The homeostasis of the cellular transcriptome depends on transcription and splicing mechanisms. Moreover, the fidelity of gene expression, essential to preserve cellular identity and function is secured by different quality control mechanisms including nonsense-mediated RNA decay (NMD). In this context, alternative splicing is coupled to NMD, and several alterations in these mechanisms leading to the accumulation of aberrant gene isoforms are known to be involved in human disease including cancer. Methods: RNA sequencing, western blotting, qPCR and co-immunoprecipitation were performed in multiple silenced culture cell lines (replicates n ≥4), primary hepatocytes and samples of animal models (Jo2, APAP, Mdr2 -/- mice, n ≥3). Results: Here we show that in animal models of liver injury and in human HCC (TCGA, non-tumoral = 50 vs. HCC = 374), the process of NMD is inhibited. Moreover, we demonstrate that the splicing factor SLU7 interacts with and preserves the levels of the NMD effector UPF1, and that SLU7 is required for correct NMD. Our previous findings demonstrated that SLU7 expression is reduced in the diseased liver, contributing to hepatocellular dedifferentiation and genome instability during disease progression. Here we build on this by providing evidence that caspases activated during liver damage are responsible for the cleavage and degradation of SLU7. Conclusions: Here we identify the downregulation of UPF1 and the inhibition of NMD as a new molecular pathway contributing to the malignant reshaping of the liver transcriptome. Moreover, and importantly, we uncover caspase activation as the mechanism responsible for the downregulation of SLU7 expression during liver disease progression, which is a new link between apoptosis and hepatocarcinogenesis. Impact and implications: The mechanisms involved in reshaping the hepatocellular transcriptome and thereby driving the progressive loss of cell identity and function in liver disease are not completely understood. In this context, we provide evidence on the impairment of a key mRNA surveillance mechanism known as nonsense-mediated mRNA decay (NMD). Mechanistically, we uncover a novel role for the splicing factor SLU7 in the regulation of NMD, including its ability to interact and preserve the levels of the key NMD factor UPF1. Moreover, we demonstrate that the activation of caspases during liver damage mediates SLU7 and UPF1 protein degradation and NMD inhibition. Our findings identify potential new markers of liver disease progression, and SLU7 as a novel therapeutic target to prevent the functional decay of the chronically injured organ.

4.
Cancer Discov ; 12(5): 1356-1377, 2022 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-35191482

RESUMEN

ABSTRACT: Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse. SIGNIFICANCE: CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171.


Asunto(s)
Neoplasias de la Mama , Células Supresoras de Origen Mieloide , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/radioterapia , Femenino , Haptoglobinas , Humanos , Células Supresoras de Origen Mieloide/metabolismo , Recurrencia Local de Neoplasia/genética , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo
6.
J Hematol Oncol ; 10(1): 23, 2017 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-28103946

RESUMEN

BACKGROUND: Activated protein C/endothelial protein C receptor (APC/EPCR) axis is physiologically involved in anticoagulant and cytoprotective activities in endothelial cells. Emerging evidence indicates that EPCR also plays a role in breast stemness and human tumorigenesis. Yet, its contribution to breast cancer progression and metastasis has not been elucidated. METHODS: Transcriptomic status of EPCR was examined in a cohort of 286 breast cancer patients. Cell growth kinetics was evaluated in control and EPCR and SPARC/osteonectin, Cwcv, and kazal-like domains proteoglycan (SPOCK1/testican 1) silenced breast cancer cells in 2D, 3D, and in co-culture conditions. Orthotopic tumor growth and lung and osseous metastases were evaluated in several human and murine xenograft breast cancer models. Tumor-stroma interactions were further studied in vivo by immunohistochemistry and flow cytometry. An EPCR-induced gene signature was identified by microarray analysis. RESULTS: Analysis of a cohort of breast cancer patients revealed an association of high EPCR levels with adverse clinical outcome. Interestingly, EPCR knockdown did not affect cell growth kinetics in 2D but significantly reduced cell growth in 3D cultures. Using several human and murine xenograft breast cancer models, we showed that EPCR silencing reduced primary tumor growth and secondary outgrowths at metastatic sites, including the skeleton and the lungs. Interestingly, these effects were independent of APC ligand stimulation in vitro and in vivo. Transcriptomic analysis of EPCR-silenced tumors unveiled an effect mediated by matricellular secreted proteoglycan SPOCK1/testican 1. Interestingly, SPOCK1 silencing suppressed in vitro 3D growth. Moreover, SPOCK1 ablation severely decreased orthotopic tumor growth and reduced bone metastatic osteolytic tumors. High SPOCK1 levels were also associated with poor clinical outcome in a subset breast cancer patients. Our results suggest that EPCR through SPOCK1 confers a cell growth advantage in 3D promoting breast tumorigenesis and metastasis. CONCLUSIONS: EPCR represents a clinically relevant factor associated with poor outcome and a novel vulnerability to develop combination therapies for breast cancer patients.


Asunto(s)
Neoplasias de la Mama/patología , Carcinoma/secundario , Receptor de Proteína C Endotelial/fisiología , Proteínas de Neoplasias/fisiología , Proteoglicanos/fisiología , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Técnicas de Cultivo de Célula , Ciclo Celular , División Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Progresión de la Enfermedad , Receptor de Proteína C Endotelial/antagonistas & inhibidores , Receptor de Proteína C Endotelial/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Organismos Libres de Patógenos Específicos , Transcriptoma , Microambiente Tumoral
7.
Nat Commun ; 8: 14294, 2017 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-28220783

RESUMEN

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers.


Asunto(s)
Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/genética , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Mutación , Oncogenes/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN , Tratamiento con ARN de Interferencia/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
8.
Cardiovasc Res ; 110(3): 309-18, 2016 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-26976620

RESUMEN

AIMS: Atrial fibrillation (AF) is a major risk factor for cardio-embolic stroke. Anticoagulant drugs are effective in preventing AF-related stroke. However, the high frequency of anticoagulant-associated major bleeding is a major concern. This study sought to identify new targets to develop safer antithrombotic therapies. METHODS AND RESULTS: Here, microarray analysis in peripheral blood cells in eight patients with AF and stroke and eight AF subjects without stroke brought to light a stroke-related gene expression pattern. HSPA1B, which encodes for heat-shock protein 70 kDa (Hsp70), was the most differentially expressed gene. This gene was down-regulated in stroke subjects, a finding confirmed further in an independent AF cohort of 200 individuals. Hsp70 knock-out mice subjected to different thrombotic challenges developed thrombosis significantly earlier than their wild-type (WT) counterparts. Remarkably, the tail bleeding time was unchanged. Accordingly, both TRC051384 and tubastatin A, i.e. two Hsp70 inducers via different pathways, delayed thrombus formation in WT mice, the tail bleeding time still being unaltered. Most interestingly, Hsp70 inducers did not increase the bleeding risk even when aspirin was concomitantly administered. Hsp70 induction was associated with an increased vascular thrombomodulin expression and higher circulating levels of activated protein C upon thrombotic stimulus. CONCLUSIONS: Hsp70 induction is a novel approach to delay thrombus formation with minimal bleeding risk, and is especially promising for treating AF patients and in other situations where there is also a major bleeding hazard.


Asunto(s)
Fibrilación Atrial/metabolismo , Enfermedades de las Arterias Carótidas/prevención & control , Proteínas HSP70 de Choque Térmico/metabolismo , Accidente Cerebrovascular/prevención & control , Trombosis/prevención & control , Animales , Aspirina/farmacología , Fibrilación Atrial/genética , Tiempo de Sangría , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Fibrinolíticos/farmacología , Perfilación de la Expresión Génica/métodos , Predisposición Genética a la Enfermedad , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas HSP70 de Choque Térmico/genética , Hemorragia/genética , Hemorragia/metabolismo , Hemorragia/prevención & control , Humanos , Ratones Noqueados , Morfolinas/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Proteína C/metabolismo , Piridinas/farmacología , Factores de Riesgo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Trombomodulina/metabolismo , Trombosis/genética , Trombosis/metabolismo , Factores de Tiempo , Regulación hacia Arriba , Urea/análogos & derivados , Urea/farmacología
9.
Mol Oncol ; 8(3): 689-703, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24593875

RESUMEN

Bone metastasis represents one of the most deleterious clinical consequences arising in the context of many solid tumors. Severe osteolysis results from tumor cell colonization of the bone compartment, a process which entails reciprocal exchange of soluble signals between tumor cells and their osseous microenvironment. Recent evidence indicates that tumor-intrinsic miRNAs are pleiotropic regulators of gene expression. But they are also frequently released in exosome-like vesicles (ELV). Yet the functional relevance of the transference of tumor-derived ELV and their miRNA cargo to the extracellular milieu during osseous colonization is unknown. Comparative transcriptomic profiling using an in vivo murine model of bone metastasis identified a repressed miRNA signature associated with high prometastatic activity. Forced expression of single miRNAs identified miR-192 that markedly appeased osseous metastasis in vivo, as shown by X-ray, bioluminescence imaging and microCT scans. Histological examination of metastatic lesions revealed impaired tumor-induced angiogenesis in vivo, an effect that was associated in vitro with decreased hallmarks of angiogenesis. Isolation and characterization of ELV by flow cytometry, Western blot analysis, transmission electron microscopy and nanoparticle tracking analysis revealed the ELV cargo enrichment in miR-192. Consistent with these findings, fluorescent labeled miR-192-enriched-ELV showed the in vitro transfer and release of miR-192 in target endothelial cells and abrogation of the angiogenic program by repression of proangiogenic IL-8, ICAM and CXCL1. Moreover, in vivo infusion of fluorescent labeled ELV efficiently targeted cells of the osseous compartment. Furthermore, treatment with miR-192 enriched ELV in a model of in vivo bone metastasis pre-conditioned osseous milieu and impaired tumor-induced angiogenesis, thereby reducing the metastatic burden and tumor colonization. Changes in the miRNA-cargo content within ELV represent a novel mechanism heavily influencing bone metastatic colonization, which is most likely relevant in other target organs. Mechanistic mimicry of this phenomenon by synthetic nanoparticles could eventually emerge as a novel therapeutic approach.


Asunto(s)
Adenocarcinoma/patología , Neoplasias Óseas/secundario , Huesos/patología , Exosomas/patología , Neoplasias Pulmonares/patología , MicroARNs/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma del Pulmón , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Huesos/metabolismo , Línea Celular Tumoral , Exosomas/genética , Exosomas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , MicroARNs/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología
10.
PLoS One ; 7(10): e47717, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23077663

RESUMEN

BACKGROUND: Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied. RESULTS: Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1. CONCLUSIONS: NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR-370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors.


Asunto(s)
Leucemia Mieloide Aguda , MicroARNs , Neurofibromina 1 , ARN Mensajero , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/metabolismo
11.
PLoS One ; 7(8): e42086, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22876301

RESUMEN

Lung cancer is a leading cause of cancer death worldwide. Several alterations in RNA metabolism have been found in lung cancer cells; this suggests that RNA metabolism-related molecules are involved in the development of this pathology. In this study, we searched for RNA metabolism-related genes that exhibit different expression levels between normal and tumor lung tissues. We identified eight genes differentially expressed in lung adenocarcinoma microarray datasets. Of these, seven were up-regulated whereas one was down-regulated. Interestingly, most of these genes had not previously been associated with lung cancer. These genes play diverse roles in mRNA metabolism: three are associated with the spliceosome (ASCL3L1, SNRPB and SNRPE), whereas others participate in RNA-related processes such as translation (MARS and MRPL3), mRNA stability (PCBPC1), mRNA transport (RAE), or mRNA editing (ADAR2, also known as ADARB1). Moreover, we found a high incidence of loss of heterozygosity at chromosome 21q22.3, where the ADAR2 locus is located, in NSCLC cell lines and primary tissues, suggesting that the downregulation of ADAR2 in lung cancer is associated with specific genetic losses. Finally, in a series of adenocarcinoma patients, the expression of five of the deregulated genes (ADAR2, MARS, RAE, SNRPB and SNRPE) correlated with prognosis. Taken together, these results support the hypothesis that changes in RNA metabolism are involved in the pathogenesis of lung cancer, and identify new potential targets for the treatment of this disease.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Adenosina Desaminasa/genética , Anciano , Anciano de 80 o más Años , Empalme Alternativo/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Línea Celular Tumoral , Análisis por Conglomerados , Biología Computacional/métodos , Femenino , Humanos , Pérdida de Heterocigocidad , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Masculino , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Pronóstico , ARN/metabolismo , Proteínas de Unión al ARN/genética
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