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1.
Mol Pharmacol ; 95(6): 638-651, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30979813

RESUMEN

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ 0 (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.


Asunto(s)
Transporte de Electrón/efectos de los fármacos , Mitocondrias/genética , Neoplasias/genética , Nitroimidazoles/farmacología , Mostazas de Fosforamida/farmacología , Análisis de Secuencia de ARN/métodos , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Células HCT116 , Humanos , Mitocondrias/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Profármacos , ARN Interferente Pequeño/farmacología
2.
Oncogene ; 37(25): 3399-3414, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29483644

RESUMEN

EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.


Asunto(s)
Glicoproteínas/metabolismo , Glicoproteínas/fisiología , Interleucina-8/metabolismo , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Neovascularización Patológica/patología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Glicoproteínas/genética , Humanos , Interleucina-8/genética , Masculino , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Ratas , Ratas Endogámicas F344 , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
3.
J Pathol ; 232(4): 391-404, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24374807

RESUMEN

EMILIN2 is an extracellular matrix (ECM) protein that exerts contradictory effects within the tumour microenvironment: it induces apoptosis in a number of tumour cells, but it also enhances tumour neo-angiogenesis. In this study, we describe a new mechanism by which EMILIN2 attenuates tumour cell viability. Based on sequence homology with the cysteine-rich domain (CRD) of the Frizzled receptors, we hypothesized that EMILIN2 could affect Wnt signalling activation and demonstrate direct interaction with the Wnt1 ligand. This physical binding leads to decreased LRP6 phosphorylation and to the down-modulation of ß-catenin, TAZ and their target genes. As a consequence, EMILIN2 negatively affects the viability, migration and tumourigenic potential of MDA-MB-231 breast cancer cells in a number of two- and three-dimensional in vitro assays. EMILIN2 does not modulate Wnt signalling downstream of the Wnt-Frizzled interaction, since it does not affect the activation of the pathway following treatment with the GSK3 inhibitors LiCl and CHIR99021. The interaction with Wnt1 and the subsequent biological effects require the presence of the EMI domain, as there is no effect with a deletion mutant lacking this domain. Moreover, in vivo experiments show that the ectopic expression of EMILIN2, as well as treatment with the recombinant protein, significantly reduce tumour growth and dissemination of cancer cells in nude mice. Accordingly, the tumour samples are characterized by a significant down-regulation of the Wnt signalling pathway. Altogether, these findings provide further evidence of the complex regulations governed by EMILIN2 in the tumour microenvironment, and they identify a key extracellular regulator of the Wnt signalling pathway.


Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Glicoproteínas/metabolismo , Vía de Señalización Wnt , Proteína Wnt1/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Desnudos , Mutación , Invasividad Neoplásica , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Carga Tumoral , Microambiente Tumoral , Proteína Wnt1/genética , beta Catenina/metabolismo
4.
Neoplasia ; 12(4): 294-304, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20360940

RESUMEN

We have recently reported that elastin microfibril interface located protein 2 (EMILIN2), an extracellular matrix (ECM) glycoprotein, triggers cell death through a direct binding to death receptors. EMILIN2 thus influences cell viability through a mechanism that is unique for an ECM molecule. In the present work, we report an additional function for this molecule. First, we identify the region responsible for the proapoptotic effects, a 90-amino acid residue-long coiled-coil fragment toward the N-terminus of the molecule. The fragment recapitulates EMILIN2 proapoptotic mechanisms. In addition, using either the full molecule or the active fragment, for the first time, we demonstrate a significant antitumoral effect in vivo, likely due to a decrease in tumor cell viability. Unexpectedly, tumors treated with EMILIN2 or the deletion mutant display a significant increase of tumor angiogenesis. In view of this novel finding, the cotreatment of the growing tumors with an antiangiogenic drug led, in most cases, to a complete regression of tumor growth. These results grant further support to recent findings that pinpoint the microenvironment as an important regulator of cell fate under both physiological and pathological conditions and disclose the possibility of using EMILIN2 fragments as potent antineoplastic tools for cancer treatment.


Asunto(s)
Glicoproteínas/fisiología , Neoplasias/patología , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/farmacología , Proteínas Reguladoras de la Apoptosis/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/farmacología , Proteínas de la Matriz Extracelular/fisiología , Glicoproteínas/química , Glicoproteínas/farmacología , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Biológicos , Fragmentos de Péptidos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Curr Cancer Drug Targets ; 8(8): 647-65, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19075588

RESUMEN

Research into mTOR, mammalian Target Of Rapamycin as an important drug target continues to be extremely interesting, both in terms of the increased molecular knowledge being acquired at the basis of various human diseases, and also for possible applications in drug cancer therapy. The mTOR signaling system plays a key role in several transduction pathways that are necessary for cell cycle progression and cellular proliferation. Drugs known as mTOR inhibitors have been included in ongoing and in recently completed cancer trials. New insights into the mTOR signaling system are helping to clarify the functionality of key mTOR components, and especially their possible role in apoptosis, angiogenesis and tumor progression. Three other molecules, already approved for therapeutic use and being commercialized (Everolimius, Temsirolimus and Zotarolimus) are added to Rapamycin (also known as Sirolimus), the parent drug of the mTOR inhibitors. Of these, only Temsirolimus is currently approved in the treatment of renal cell carcinoma, while the others are approved for organ transplant rejection and coronary artery restenosis. There are at least 10 other molecules currently under development for clinical and preclinical studies. This review offers an updated synopsis of the mTOR signaling system, in particular as regards relevant aspects of cancer research, looks at the known mTOR inhibitors and gives a systematic vision of current trials for each individual molecule subject to clinical investigation.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Animales , Antineoplásicos/uso terapéutico , Diseño de Drogas , Humanos , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR
6.
Connect Tissue Res ; 49(3): 203-6, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18661343

RESUMEN

Extracellular matrix (ECM) is an essential component of the stromal microenvironment both from a structural and a functional point of view. The ECM functions as a scaffold for tissue organization and regulates growth factors and chemokines availability thus contributing to maintain tissue homeostasis. Attachment of cells to ECM is essential to support cell survival, growth, and proliferation, and the lack of these interactions can trigger a type of cell death named anoikis. Several studies point out that alterations of ECM composition are often responsible of many pathological conditions such as cancer, of which it has been demonstrated to be occasionally the main promoter. ECM does not always represent a prosurvival stimulus; among the different array of ECM molecules a set of proteins can negatively affect cell viability and are thought to play an important role in tumor progression. For this reason attention has been focused on these molecules as potential tools or targets for therapy.


Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Animales , Anoicis/fisiología , Homeostasis , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas/metabolismo
7.
Matrix Biol ; 27(2): 96-106, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17988845

RESUMEN

The informational spectrum method (ISM) is a virtual spectroscopy method for the fast analysis of potential protein-protein relationships. By applying the ISM approach to the GeneBank protein database the vascular proteins EMILIN1 (Elastin Microfibril Interface Located ProteIN), EMILIN2, MMN1, and MMN2 were identified as additional anthrax PA antigen interacting molecules. This virtual molecular interaction was formally proven by solid phase assays using recombinant proteins. The interaction is independent of the presence of divalent cations and does not involve PA aspartic residue at 683, a critical residue in receptor binding. In fact, the D683A point mutation fully prevented the cell intoxication ability of PA in the presence of Lethal Factor, but it was fully ineffective on the binding of mutated PA to EMILIN1 and EMILIN2. The ISM approach also led to the identification of the potential interaction sites between PA and EMILINs. A PA mutant with a deletion at residue D425 and solid phase protein-protein interaction studies as well as deletion mutant of EMILIN2 confirmed the hypothesized interaction site. Our findings imply that the PA-cell surface receptor interaction is not likely to provide the full explanation for the vascular lesions and prominent hemorrhages that follow Bacillus anthracis infection and spreading and call into play vascular associated proteins such as EMILINs as potential inhibitory proteins.


Asunto(s)
Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos Bacterianos/farmacología , Antígenos de Superficie/química , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/farmacología , Sitios de Unión , Proteínas Sanguíneas/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Biología Computacional/métodos , Bases de Datos Genéticas , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Péptidos , Proteínas Recombinantes/metabolismo
8.
Mol Cell Biol ; 27(20): 7176-87, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17698584

RESUMEN

Elastin microfibril interface-located proteins (EMILINs) constitute a family of extracellular matrix (ECM) glycoproteins characterized by the presence of an EMI domain at the N terminus and a gC1q domain at the C terminus. EMILIN1, the archetype molecule of the family, is involved in elastogenesis and hypertension etiology, whereas the function of EMILIN2 has not been resolved. Here, we provide evidence that the expression of EMILIN2 triggers the apoptosis of different cell lines. Cell death depends on the activation of the extrinsic apoptotic pathway following EMILIN2 binding to the TRAIL receptors DR4 and, to a lesser extent, DR5. Binding is followed by receptor clustering, colocalization with lipid rafts, death-inducing signaling complex assembly, and caspase activation. The direct activation of death receptors by an ECM molecule that mimics the activity of the known death receptor ligands is novel. The knockdown of EMILIN2 increases transformed cell survival, and overexpression impairs clonogenicity in soft agar and three-dimensional growth in natural matrices due to massive apoptosis. These data demonstrate an unexpected direct and functional interaction of an ECM constituent with death receptors and discloses an additional mechanism by which ECM cues can negatively affect cell survival.


Asunto(s)
Apoptosis/fisiología , Glicoproteínas/metabolismo , Transducción de Señal/fisiología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Transformación Celular Neoplásica , Activación Enzimática , Fibroblastos/citología , Fibroblastos/fisiología , Glicoproteínas/genética , Humanos , Precursores de Proteínas/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo
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