RESUMEN
H-1 parvovirus (H-1PV) is a promising anticancer therapy. However, in-depth understanding of its life cycle, including the host cell factors needed for infectivity and oncolysis, is lacking. This understanding may guide the rational design of combination strategies, aid development of more effective viruses, and help identify biomarkers of susceptibility to H-1PV treatment. To identify the host cell factors involved, we carry out siRNA library screening using a druggable genome library. We identify one crucial modulator of H-1PV infection: laminin γ1 (LAMC1). Using loss- and gain-of-function studies, competition experiments, and ELISA, we validate LAMC1 and laminin family members as being essential to H-1PV cell attachment and entry. H-1PV binding to laminins is dependent on their sialic acid moieties and is inhibited by heparin. We show that laminins are differentially expressed in various tumour entities, including glioblastoma. We confirm the expression pattern of laminin γ1 in glioblastoma biopsies by immunohistochemistry. We also provide evidence of a direct correlation between LAMC1 expression levels and H-1PV oncolytic activity in 59 cancer cell lines and in 3D organotypic spheroid cultures with different sensitivities to H-1PV infection. These results support the idea that tumours with elevated levels of γ1 containing laminins are more susceptible to H-1PV-based therapies.
Asunto(s)
Parvovirus H-1/metabolismo , Laminina/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Virus Oncolíticos/metabolismo , Acoplamiento Viral , Internalización del Virus , Animales , Línea Celular Tumoral , Glioblastoma/patología , Glioblastoma/terapia , Glioblastoma/virología , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Laminina/genética , Ratones Endogámicos NOD , Ratones SCID , Viroterapia Oncolítica/métodos , Unión Proteica , Interferencia de ARN , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The SHOX gene encodes for a transcription factor important for normal bone development. Mutations in the gene are associated with idiopathic short stature and are responsible for the growth failure and skeletal defects found in the majority of patients with Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia. SHOX is expressed in growth plate chondrocytes where it is supposed to modulate the proliferation, differentiation and cell death of these cells. Supporting this hypothesis, in vitro studies have shown that SHOX expression induces cell cycle arrest and apoptosis in both transformed and primary cells. In this study, we further characterized the cell death mechanisms triggered by SHOX and compared them with the effects induced by one clinically relevant mutant form of SHOX, detected in LWD patients (SHOX R153L) and a SHOX C-terminally truncated version (L185X). We show that SHOX expression in U2OS osteosarcoma cells leads to oxidative stress that, in turn, induces lysosomal membrane rupture with release of active cathepsin B to the cytosol and subsequent activation of the intrinsic apoptotic pathway characterized by mitochondrial membrane permeabilization and caspase activation. Importantly, cells expressing SHOX R153L or L185X did not display any of these features. Given the fact that many of the events observed in SHOX-expressing cells also characterize the complex cell death process occurring in the growth plate during endochondral ossification, our findings further support the hypothesis that SHOX may play a central role in the regulation of the cell death pathways activated during long bone development.
Asunto(s)
Trastornos del Crecimiento/genética , Proteínas de Homeodominio/metabolismo , Lisosomas/genética , Osteocondrodisplasias/genética , Osteosarcoma/genética , Estrés Oxidativo , Apoptosis , Caspasas/metabolismo , Catepsina B/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Trastornos del Crecimiento/patología , Placa de Crecimiento/metabolismo , Humanos , Lisosomas/metabolismo , Mutación , Osteocondrodisplasias/patología , Osteosarcoma/metabolismo , Proteína de la Caja Homeótica de Baja EstaturaRESUMEN
The rat parvovirus H-1PV has oncolytic and tumour-suppressive properties potentially exploitable in cancer therapy. This possibility is being explored and results are encouraging, but it is necessary to improve the oncotoxicity of the virus. Here we show that this can be achieved by co-treating cancer cells with H-1PV and histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA). We demonstrate that these agents act synergistically to kill a range of human cervical carcinoma and pancreatic carcinoma cell lines by inducing oxidative stress, DNA damage and apoptosis. Strikingly, in rat and mouse xenograft models, H-1PV/VPA co-treatment strongly inhibits tumour growth promoting complete tumour remission in all co-treated animals. At the molecular level, we found acetylation of the parvovirus nonstructural protein NS1 at residues K85 and K257 to modulate NS1-mediated transcription and cytotoxicity, both of which are enhanced by VPA treatment. These results warrant clinical evaluation of H-1PV/VPA co-treatment against cervical and pancreatic ductal carcinomas.
Asunto(s)
Carcinoma/terapia , Virus Oncolíticos/fisiología , Parvovirus/fisiología , Ácido Valproico/farmacología , Animales , Apoptosis/efectos de los fármacos , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Células HeLa , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Estrés Oxidativo/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Parvovirus/metabolismo , Ratas , Ratas Desnudas , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Ácido Valproico/uso terapéuticoRESUMEN
The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α(v)ß(3) and α(v)ß(5) integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α(v)ß(5) integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.
Asunto(s)
Proteínas de la Cápside/genética , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Parvovirus/genética , Animales , Células CHO , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Cricetinae , Ingeniería Genética , Humanos , Modelos Moleculares , Neoplasias/virología , Virus Oncolíticos/química , Virus Oncolíticos/fisiología , Infecciones por Parvoviridae/virología , Parvovirus/química , Parvovirus/fisiología , Ratas , Replicación ViralRESUMEN
Short stature due to SHOX deficiency represents a common congenital form of growth failure and is involved in the aetiology of 'idiopathic' short stature and the growth deficits and skeletal anomalies in Leri-Weill, Langer and Turner syndromes. Although much is known on the clinical and molecular aspects of SHOX haploinsufficiency, the integration of SHOX in the signalling pathways regulating bone growth is currently not defined. Here we identify NPPB encoding the natriuretic peptide, BNP, a well-known cardiac and natriuretic peptide hormone, as a transcriptional target of SHOX. The ability of SHOX to transactivate the NPPB endogenous promoter was demonstrated in luciferase reporter assays using serial deletions of the NPPB promotor region. Binding of SHOX to the NPPB promoter was also demonstrated in vivo by chromatin fixation and immunoprecipitation. We also demonstrate the lack of promoter activation in two SHOX mutants from patients with Leri-Weill syndrome. In addition, immunohistochemical analysis of human growth plate sections showed for the first time a co-expression of BNP and SHOX in late proliferative and hypertrophic chondrocytes. Together these data strongly suggest that BNP represents a direct target of SHOX.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Homeodominio/fisiología , Péptido Natriurético Encefálico/genética , Adolescente , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Línea Celular Tumoral , Condrocitos/metabolismo , Condrosarcoma/genética , Condrosarcoma/patología , Femenino , Placa de Crecimiento/metabolismo , Placa de Crecimiento/patología , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Péptido Natriurético Encefálico/metabolismo , Osteosarcoma/genética , Osteosarcoma/patología , Elementos de Respuesta , Proteína de la Caja Homeótica de Baja EstaturaRESUMEN
Mutations within the homeobox SHOX gene have been associated with short stature and the skeletal deformities found in Léri-Weill, Turner and Langer syndromes implying an involvement of SHOX in growth and bone formation. Despite its clinical significance, the precise role of SHOX and the mechanisms that modulate its functions remain unknown. We reported previously that SHOX is a nuclear protein that specifically binds DNA and acts as a transcriptional activator. We have shown that ectopic expression of SHOX leads to cell-cycle arrest and apoptosis in osteosarcoma and primary cells. To further characterize SHOX, we investigated whether the protein could be a target for phosphorylation. Here, we report that SHOX is phosphorylated exclusively on serine residues in vivo. Two-dimensional phospho-peptide mapping showed that SHOX is phosphorylated to various extents on multiple sites. Site-directed mutagenesis demonstrated that serine 106 is the major SHOX phosphorylation site. We show also that casein kinase II phosphorylates SHOX on serine 106 efficiently in vitro and specific casein kinase II inhibitors reduce SHOX phosphorylation strongly in vivo. Finally, we provide evidence that phosphorylation may play an important role in modulating SHOX biological activities, since a S106A SHOX mutant, defective in phosphorylation, does not activate transcription and fails to induce cell-cycle arrest and apoptosis.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proteínas de Homeodominio/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Proteína de la Caja Homeótica de Baja Estatura , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Haploinsufficiency of the short stature homeobox gene SHOX has been found in patients with idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). In addition to complete gene deletions and nonsense mutations, several missense mutations have been identified in both patient groups, leading to amino acid substitutions in the SHOX protein. The majority of missense mutations were found to accumulate in the region encoding the highly conserved homeodomain of the paired-like type. In this report, we investigated nine different amino acid exchanges in the homeodomain of SHOX patients with ISS and LWD. We were able show that these mutations cause an alteration of the biological function of SHOX by loss of DNA binding, reduced dimerization ability, and/or impaired nuclear translocation. Additionally, one of the mutations (c.458G>T, p.R153L) is defective in transcriptional activation even though it is still able to bind to DNA, dimerize, and translocate to the nucleus. Thus, we demonstrate that single missense mutations in the homeodomain fundamentally impair SHOX key functions, thereby leading to the phenotype observed in patients with LWD and ISS.
Asunto(s)
Estatura/genética , Núcleo Celular/metabolismo , ADN/metabolismo , Trastornos del Crecimiento/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Mutación/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Ciclo Celular , Dimerización , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Humanos , Datos de Secuencia Molecular , Mutación Missense/genética , Proteína de la Caja Homeótica de Baja Estatura , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.